Fc-region variants with improved protein A-binding

ABSTRACT

Herein is reported a polypeptide comprising a first polypeptide and a second polypeptide each comprising in N-terminal to C-terminal direction at least a portion of an immunoglobulin hinge region, which comprises one or more cysteine residues, an immunoglobulin CH2-domain and an immunoglobulin CH3-domain, wherein the first, the second, or the first and the second polypeptide comprise the mutation Y436A (numbering according to the EU index).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/210,464, filed Jul. 14, 2016, which is a continuation ofInternational Application No. PCT/EP2015/050389, having an internationalfiling date of Jan. 12, 2015, the entire contents of which areincorporated herein by reference, and which claims benefit to EuropeanApplication No. 14151322.6, filed Jan. 15, 2014, and EuropeanApplication No. 14165924.3, filed Apr. 25, 2014.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted viaEFS-Web and hereby incorporated by reference in its entirety. Said ASCIIcopy, created on Jan. 8, 2019, is namedP31954-US-1_Sequence_Listing.txt, and is 308,687 bytes in size.

FIELD OF THE INVENTION

Herein are reported IgG Fc-regions that have been modified with respectto their purification properties.

BACKGROUND OF THE INVENTION

The demand for cost efficient production processes has led to thenecessity of optimization of the downstream purification, including oneor more affinity chromatography steps. Larger volumes to be processedand harder requirements for the cleaning-in-place (CIP) protocols aresome of the features that need to be solved (Hober, S., J. Chrom. B. 848(2007) 40-47).

The purification of monoclonal antibodies by means of selectiveFc-region affinity ligands is the most promising methodology for thelarge-scale production of therapeutic monoclonal antibodies. In fact,this procedure does not require establishing any interaction with theantigen specific part of the antibody, i.e. the Fab domain, which is,thus, left intact and can retain its properties (see Salvalaglio, M., etal., J. Chrom. A 1216 (2009) 8678-8686).

Due to its selectiveness, an affinity-purification step is employedearly in the purification chain and thereby the number of successiveunit operations can be reduced (see Hober supra; MacLennan, J.,Biotechnol. 13 (1995) 1180; Harakas, N. K., Bioprocess Technol. 18(1994) 259).

The ligands most adopted to bind selectively IgG are Staphylococcalprotein A and protein G, which are able to establish highly selectiveinteractions with the Fc-region of most IgGs in a region known as“consensus binding site” (CBS) (DeLano, W. L., et al., Science 287(2000) 1279), which is located at the hinge region between the CH2 andCH3 domains of the Fc-region.

Staphylococcal protein A (SPA) is a cell wall associated protein domainexposed on the surface of the Gram-positive bacterium Staphylococcusaureus. SPA has high affinity to IgG from various species, for instancehuman, rabbit and guinea pig IgG but only weak interaction with bovineand mouse IgG (see the following Table) (see Hober supra; Duhamel, R.C., et al., J. Immunol. Methods 31 (1979) 211; Björk, L. and Kronvall,G., Immunol. J. 133 (1984) 969; Richman, D. D., et al., J. Immunol. 128(1982) 2300; Amersham Pharmacia Biotech, Handbook, Antibody Purification(2000)).

species subclass protein A binding human IgG1 ++ IgG2 ++ IgG3 −− IgG4 ++IgA variable IgD − IgM variable rabbit no distinction ++ guinea pig IgG1++ IgG2 ++ bovine + mouse IgG1 + IgG2a ++ IgG2b + IgG3 + IgM variablechicken IgY − ++: strong binding/+: medium binding/−: weak or nointeraction

The heavy chain hinge-region between the CH2 and CH3 domains of IgG isable to bind several proteins beyond protein A, such as the neonatal Fcreceptor (FcRn) (see DeLano and Salvalaglio supra).

The SPA CBS comprehends a hydrophobic pocket on the surface of theantibody. The residues composing the IgG CBS are Ile 253, Ser 254, Met252, Met 423, Tyr 326, His 435, Asn 434, His 433, Arg 255 and Glu 380(numbering of the IgG heavy chain residues according to the Kabat EUindex numbering system). The charged amino acids (Arg 255, Glu 380) areplaced around a hydrophobic knob formed by Ile 253 and Ser 254. This(can) result in the establishment of polar and hydrophilic interactions(see Salvalaglio supra).

In general, the protein A-IgG interaction can be described using twomain binding sites: the first is positioned in the heavy chain CH2domain and is characterized by hydrophobic interactions between Phe 132,Leu 136, Ile 150 (of protein A) and the IgG hydrophobic knob constitutedby Ile 253 and Ser 254, and by one electrostatic interaction between Lys154 (protein A) and Thr 256 (IgG). The second site is located in theheavy chain CH3 domain and is dominated by electrostatic interactionsbetween Gln 129 and Tyr 133 (protein A) and His 433, Asn 434, and His435 (IgG) (see Salvalaglio supra).

Lindhofer, H., et al. (J. Immunol. 155 (1995) 219-225) reportpreferential species-restricted heavy/light chain pairing in rat/mousequadromas.

Jedenberg, L., et al. (J. Immunol. Meth. 201 (1997) 25-34) reported thatSPA-binding analyses of two Fc variants (Fc13 and Fc31, each containingan isotypic dipeptide substitution from the respective other isotype)showed that Fc1 and Fc31 interact with SPA, while Fc3 and Fc13 lackdetectable SPA binding. The rendered SPA binding of the Fc-regionvariant Fc31 is concluded to result from the introduced dipeptidesubstitution R435H and F436Y.

Today, the focus with respect to therapeutic monoclonal antibodies is onthe generation and use of bispecific or even multispecific antibodiesspecifically binding to two or more targets (antigens).

The basic challenge in generating multispecific heterodimeric IgGantibodies from four antibody chains (two different heavy chains and twodifferent light chains) in one expression cell line is the so-calledchain association issue (see Klein, C., et al., mAbs 4 (2012) 653-663).The required use of different chains as the left and the right arm ofthe multispecific antibody leads to antibody mixtures upon expression inone cell: the two heavy chains are able to (theoretically) associate infour different combinations (two thereof are identical), and each ofthose can associate in a stochastic manner with the light chains,resulting in 2⁴ (=a total of 16) theoretically possible chaincombinations. Of the 16 theoretically possible combinations ten can befound of which only one corresponds to the desired functional bispecificantibody (De Lau, W. B., et al., J. Immunol. 146 (1991) 906-914). Thedifficulties in isolating this desired bispecific antibody out ofcomplex mixtures and the inherent poor yield of 12.5% at a theoreticalmaximum make the production of a bispecific antibody in one expressioncell line extremely challenging.

To overcome the chain association issue and enforce the correctassociation of the two different heavy chains, in the late 1990s Carteret al. from Genentech invented an approach termed “knobs-into-holes”(KiH) (see Carter, P., J. Immunol. Meth. 248 (2001) 7-15; Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681; Zhu, Z., et al., Prot.Sci. 6 (1997) 781-788; Ridgway, J. B., et al., Prot. Eng. 9 (1996)617-621; Atwell, S., et al., J. Mol. Biol. 270(1997) 26-35; and U.S.Pat. No. 7,183,076). Basically, the concept relies on modifications ofthe interface between the two CH3 domains of the two heavy chains of anantibody where most interactions occur. A bulky residue is introducedinto the CH3 domain of one antibody heavy chain and acts similarly to akey (“knob”). In the other heavy chain, a “hole” is formed that is ableto accommodate this bulky residue, mimicking a lock. The resultingheterodimeric Fc-region can be further stabilized by theintroduction/formation of artificial disulfide bridges. Notably, all KiHmutations are buried within the CH3 domains and not “visible” to theimmune system. In addition, properties of antibodies with KiH mutationssuch as (thermal) stability, FcγR binding and effector functions (e.g.,ADCC, FcRn binding) and pharmacokinetic (PK) behavior are not affected.

Correct heavy chain association with heterodimerization yields above 97%can be achieved by introducing six mutations: S354C, T366W in the “knob”heavy chain and Y349C, T366S, L368A, Y407V in the “hole” heavy chain(see Carter supra; numbering of the residues according to the Kabat EUindex numbering system). While hole-hole homodimers may occur, knob-knobhomodimers typically are not observed. Hole-hole dimers can either bedepleted by selective purification procedures or by procedures asoutlined below.

While the issue of random heavy chain association has been addressed,also correct light chain association has to be ensured. Similar to theKiH CH3 domain approach, efforts have been undertaken to investigateasymmetric light chain-heavy chain interactions that might ultimatelylead to full bispecific IgGs.

Roche recently developed the CrossMab approach as a possibility toenforce correct light chain pairing in bispecific heterodimeric IgGantibodies when combining it with the KiH technology (see Klein supra;Schaefer. W., et al., Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192;Cain, C., SciBX 4 (2011) 1-4). This allows the generation of bispecificor even multispecific antibodies in a generic fashion. In this format,one arm of the intended bispecific antibody is left untouched. In thesecond arm, the whole Fab region, or the VH-VL domains or the CH1-CLdomains are exchanged by domain crossover between the heavy and lightchain. As a consequence, the newly formed “crossed” light chain does notassociate with the (normal, i.e. not-crossed) heavy chain Fab region ofthe other arm of the bispecific antibody any longer. Thus, the correct“light chain” association can be enforced by this minimal change indomain arrangement (see Schaefer supra).

Zhu et al. introduced several sterically complementary mutations, aswell as disulfide bridges, in the two VL/VH interfaces of diabodyvariants. When the mutations VL Y87A/F98M and VH V37F/L45W wereintroduced into the anti-p185HER2 VL/VH interface, a heterodimericdiabody was recovered with >90% yield while maintaining overall yieldand affinity compared with the parental diabody (see Zhu supra).

Researchers from Chugai have similarly designed bispecific diabodies byintroduction of mutations into the VH-VL interfaces (mainly conversionof Q39 in VH and Q38 in VL to charged residues) to foster correct lightchain association (WO 2006/106905; Igawa, T., et al., Prot. Eng. Des.Sel. 23 (2010) 667-677).

In WO2011097603 a common light chain mouse is reported.

In WO2010151792 a bispecific antibody format providing ease of isolationis provided, comprising immunoglobulin heavy chain variable domains thatare differentially modified, i.e. heterodimeric, in the CH3 domain,wherein the differential modifications are non-immunogenic orsubstantially non-immunogenic with respect to the CH3 modifications, andat least one of the modifications results in a differential affinity forthe bispecific antibody for an affinity reagent such as protein A, andthe bispecific antibody is isolable from a disrupted cell, from medium,or from a mixture of antibodies based on its affinity for protein A.

The neonatal Fc-receptor (FcRn) is important for the metabolic fate ofantibodies of the IgG class in vivo. The FcRn functions to salvage IgGfrom the lysosomal degradation pathway, resulting in reduced clearanceand increased half-life. It is a heterodimeric protein consisting of twopolypeptides: a 50 kDa class I major histocompatibility complex-likeprotein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds withhigh affinity to the CH2-CH3 portion of the Fc-region of an antibody ofthe class IgG. The interaction between an antibody of the class IgG andthe FcRn is pH dependent and occurs in a 1:2 stoichiometry, i.e. one IgGantibody molecule can interact with two FcRn molecules via its two heavychain Fc-region polypeptides (see e.g. Huber, A. H., et al., J. Mol.Biol. 230 (1993) 1077-1083).

Thus, the in vitro FcRn binding properties/characteristics of an IgG areindicative of its in vivo pharmacokinetic properties in the bloodcirculation.

In the interaction between the FcRn and the Fc-region of an antibody ofthe IgG class different amino acid residues of the heavy chain CH2- andCH3-domain are participating.

Different mutations that influence the FcRn binding and therewith thehalf-life in the blood circulation are known. Fc-region residuescritical to the mouse Fc-region-mouse FcRn interaction have beenidentified by site-directed mutagenesis (see e.g. Dall'Acqua, W. F., etal. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434and H435 (numbering according to Kabat EU index numbering system) areinvolved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26(1996) 2533-2536; Firan, M., et al., Int. Immunol. 13 (2001) 993-1002;Kim, J. K., et al., Eur. J. Immunol. 24 (1994) 542-548). Residues I253,H310, and H435 were found to be critical for the interaction of humanFc-region with murine FcRn (Kim, J. K., et al., Eur. J. Immunol. 29(1999) 2819-2885).

Methods to increase Fc-region (and likewise IgG) binding to FcRn havebeen performed by mutating various amino acid residues in the Fc-region:Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433,and Asn 434 (see Kuo, T. T., et al., J. Clin. Immunol. 30 (2010)777-789; Ropeenian, D. C., et al., Nat. Rev. Immunol. 7 (2007) 715-725).

The combination of the mutations M252Y, S254T, T256E have been describedby Dall'Acqua et al. to improve FcRn binding by protein-proteininteraction studies (Dall'Acqua, W. F., et al. J. Biol. Chem. 281 (2006)23514-23524). Studies of the human Fc-region-human FcRn complex haveshown that residues I253, S254, H435 and Y436 are crucial for theinteraction (Firan, M., et al., Int. Immunol. 13 (2001) 993-1002;Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung,Y. A., et al. (J. Immunol. 182 (2009) 7667-7671) various mutants ofresidues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 havebeen reported and examined.

In WO 2014/006217 dimeric proteins with triple mutations are reported.Crystal structure at 2.8 Angstrom of an FcRn/heterodimeric Fc complexregarding the mechanism of pH-dependent binding was reported by Martin,W., et al. (Mol. Cell. 7 (2001) 867-877). In U.S. Pat. No. 6,277,375,immunoglobulin like domains with increased half-lives are reported.Shields, R. L., et al., reported high resolution mapping of the bindingsite on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, andFcRn and design of IgG1 variants with improved binding to the Fc gamma R(Biochem. Mol. Biol. 276 (2001) 6591-6604). The delineation of the aminoacid residues involved in transcytosis and catabolism of mouse IgG1 wasreported by Medesan, C., et al. (J. Immunol. 158 (1997) 2211-2217). InUS 2010/0272720 antibody fusion proteins with a modified FcRn bindingsite are reported. The production of heterodimeric proteins is reportedin WO 2013/060867. Qiao, S.-W., et al. reported the dependence ofantibody-mediated presentation of antigen on FcRn (Proc. Natl. Acad.Sci. USA 105 (2008) 9337-9342.

SUMMARY OF THE INVENTION

Herein are reported variant Fc-regions that specifically bind toStaphylococcus protein A and that do not bind to human FcRn. Thesevariant Fc-regions contain specific amino acid mutations in the CH2- andCH3-domain. It has been found that these mutations, when used either inthe hole chain or the knob chain of a heterodimeric Fc-region allow forthe purification of the heterodimeric Fc-region, i.e. the separation ofa heterodimeric Fc-region from a homodimeric Fc-region.

One aspect as reported herein is the use of the mutation Y436A forincreasing the binding of a (dimeric) Fc-region polypeptide to proteinA.

One aspect as reported herein is a (dimeric) polypeptide comprising

-   -   a first polypeptide comprising in N-terminal to C-terminal        direction at least a portion of an immunoglobulin hinge region,        which comprises one or more cysteine residues, an immunoglobulin        CH2-domain and an immunoglobulin CH3-domain, and a second        polypeptide comprising in N-terminal to C-terminal direction at        least a portion of an immunoglobulin hinge region, which        comprises one or more cysteine residues, an immunoglobulin        CH2-domain and an immunoglobulin CH3-domain,    -   wherein the first, the second, or the first and the second        polypeptide comprise the mutation Y436A (numbering according to        the Kabat EU index numbering system), and    -   wherein the first polypeptide and the second polypeptide are        connected by one or more disulfide bridges in the at least a        portion of an immunoglobulin hinge region.

In one embodiment the first and the second polypeptide comprise themutation Y436A.

In one embodiment the (dimeric) polypeptide does not specifically bindto the human FcRn and does specifically bind to Staphylococcal proteinA.

In one embodiment the (dimeric) polypeptide is a homodimericpolypeptide.

In one embodiment the (dimeric) polypeptide is a heterodimericpolypeptide.

In one embodiment the first polypeptide further comprises the mutationsY349C, T366S, L368A and Y407V (“hole”) and the second polypeptidecomprises the mutations S354C and T366W (“knob”).

In one embodiment the first polypeptide further comprises the mutationsS354C, T366S, L368A and Y407V (“hole”) and the second polypeptidecomprises the mutations Y349C and T366W (“knob”).

In one embodiment

-   -   i) the first and the second polypeptide each comprise the        mutations H310A, H433A and Y436A, or    -   ii) the first and the second polypeptide each further comprise        the mutations L251D, L314D and L432D, or    -   iii) the first and the second polypeptide each further comprise        the mutations L251S, L314S and L432S, or    -   iv) the first polypeptide comprises the mutations I253A, H310A        and H435A and the second polypeptide comprises the mutations        H310A, H433A and Y436A, or    -   v) the first polypeptide comprises the mutations I253A, H310A        and H435A and the second polypeptide comprises the mutations        L251D, L314D and L432D, or    -   vi) the first polypeptide comprises the mutations I253A, H310A        and H435A and the second polypeptide comprises the mutations        L251S, L314S and L432S.

In one embodiment the immunoglobulin hinge region, the immunoglobulinCH2-domain, and the immunoglobulin CH3-domain of the first and thesecond polypeptide are of the human IgG1 subclass. In one embodiment thefirst polypeptide and the second polypeptide each further comprise themutations L234A and L235A. In one embodiment the first polypeptide andthe second polypeptide each further comprise the mutation P329G. In oneembodiment the first polypeptide and the second polypeptide each furthercomprise the mutations L234A, L235A and P329G.

In one embodiment the immunoglobulin hinge region, the immunoglobulinCH2-domain and the immunoglobulin CH3-domain of the first and the secondpolypeptide are of the human IgG2 subclass. In one embodiment the firstpolypeptide and the second polypeptide each further comprise themutations H268Q, V309L, A330S and P331S.

In one embodiment the immunoglobulin hinge region, the immunoglobulinCH2-domain and the immunoglobulin CH3-domain of the first and the secondpolypeptide are of the human IgG2 subclass. In one embodiment the firstpolypeptide and the second polypeptide each further comprise themutations V234A, G237A, P238S, H268A, V309L, A330S and P331S.

In one embodiment the immunoglobulin hinge region, the immunoglobulinCH2-domain and the immunoglobulin CH3-domain of the first and the secondpolypeptide are of the human IgG4 subclass. In one embodiment the firstpolypeptide and the second polypeptide each further comprise themutations S228P and L235EA. In one embodiment the first polypeptide andthe second polypeptide each further comprise the mutation P329G. In oneembodiment the first polypeptide and the second polypeptide each furthercomprise the mutations S228P, L235E and P329G.

In one embodiment the immunoglobulin hinge region, the immunoglobulinCH2-domain and the immunoglobulin CH3-domain of the first and the secondpolypeptide are of the human IgG4 subclass. In one embodiment the firstpolypeptide and the second polypeptide each further comprise themutations S228P, L234A and L235A. In one embodiment the firstpolypeptide and the second polypeptide each further comprise themutation P329G. In one embodiment the first polypeptide and the secondpolypeptide each further comprise the mutations S228P, L234A, L235A andP329G.

In one embodiment the (dimeric) polypeptide is an Fc-region fusionpolypeptide.

In one embodiment the (dimeric) polypeptide is an (full length)antibody.

In one embodiment the (full length) antibody is a monospecific antibody.In one embodiment the monospecific antibody is a monovalent monospecificantibody. In one embodiment the monospecific antibody is a bivalentmonospecific antibody.

In one embodiment the (full length) antibody is a bispecific antibody.In one embodiment the bispecific antibody is a bivalent bispecificantibody. In one embodiment the bispecific antibody is a tetravalentbispecific antibody.

In one embodiment the (full length) antibody is a trispecific antibody.In one embodiment the trispecific antibody is a trivalent trispecificantibody. In one embodiment the trispecific antibody is a tetravalenttrispecific antibody.

One aspect as reported herein is method of treatment of a patientsuffering from ocular vascular diseases by administering a (dimeric)polypeptide or an antibody as reported herein to a patient in the needof such treatment.

One aspect as reported herein is a (dimeric) polypeptide or an antibodyas reported herein for intravitreal application.

One aspect as reported herein is a (dimeric) polypeptide or an antibodyas reported herein for use as a medicament.

One aspect as reported herein is a (dimeric) polypeptide or an antibodyas reported herein for the treatment of vascular eye diseases.

One aspect as reported herein is a pharmaceutical formulation comprisinga (dimeric) polypeptide or an antibody as reported herein and optionallya pharmaceutically acceptable carrier.

For using an antibody that targets/binds to antigens not only present inthe eye but also in the remaining body, a short systemic half-live afterpassage of the blood-ocular-barrier from the eye into the blood isbeneficial in order to avoid systemic side effects.

Additionally an antibody that specifically binds to ligands of areceptor is only effective in the treatment of eye-diseases if theantibody-antigen complex is removed from the eye, i.e. the antibodyfunctions as a transport vehicle for receptor ligands out of the eye andthereby inhibits receptor signaling.

It has been found by the current inventors that an antibody comprisingan Fc-region that does not bind to the human neonatal Fc-receptor, i.e.a (dimeric) polypeptide as reported herein, is transported across theblood-ocular barrier. This is surprising as the antibody does not bindto human FcRn although binding to FcRn is considered to be required fortransport across the blood-ocular-barrier.

One aspect as reported herein is the use of a (dimeric) polypeptide oran antibody as reported herein for the transport of a soluble receptorligand from the eye over the blood-ocular-barrier into the bloodcirculation.

One aspect as reported herein is the use of a (dimeric) polypeptide oran antibody as reported herein for the removal of one or more solublereceptor ligands from the eye.

One aspect as reported herein is the use of a (dimeric) polypeptide oran antibody as reported herein for the treatment of eye diseases,especially of ocular vascular diseases.

One aspect as reported herein is the use of a (dimeric) polypeptide oran antibody as reported herein for the transport of one or more solublereceptor ligands from the intravitreal space to the blood circulation.

One aspect as reported herein is a (dimeric) polypeptide or an antibodyas reported herein for use in treating an eye disease.

One aspect as reported herein is a (dimeric) polypeptide or an antibodyas reported herein for use in the transport of a soluble receptor ligandfrom the eye over the blood-ocular-barrier into the blood circulation.

One aspect as reported herein is a (dimeric) polypeptide or an antibodyas reported herein for use in the removal of one or more solublereceptor ligands from the eye.

One aspect as reported herein is a (dimeric) polypeptide or an antibodyas reported herein for use in treating eye diseases, especially ocularvascular diseases.

One aspect as reported herein is a (dimeric) polypeptide or an antibodyas reported herein for use in the transport of one or more solublereceptor ligands from the intravitreal space to the blood circulation.

One aspect as reported herein is a method of treating an individualhaving an ocular vascular disease comprising administering to theindividual an effective amount of a (dimeric) polypeptide or an antibodyas reported herein.

One aspect as reported herein is a method for transporting a solublereceptor ligand from the eye over the blood-ocular-barrier into theblood circulation in an individual comprising administering to theindividual an effective amount of a (dimeric) polypeptide or an antibodyas reported herein to transport a soluble receptor ligand from the eyeover the blood-ocular-barrier into the blood circulation.

One aspect as reported herein is a method for the removal of one or moresoluble receptor ligands from the eye in an individual comprisingadministering to the individual an effective amount of a (dimeric)polypeptide or an antibody as reported herein to remove one or moresoluble receptor ligands from the eye.

One aspect as reported herein is a method for the transport of one ormore soluble receptor ligands from the intravitreal space to the bloodcirculation in an individual comprising administering to the individualan effective amount of a (dimeric) polypeptide or an antibody asreported herein to transport one or more soluble receptor ligands fromthe intravitreal space to the blood circulation.

One aspect as reported herein is a method for transporting a solublereceptor ligand from the intravitreal space or the eye over theblood-ocular-barrier into the blood circulation in an individualcomprising administering to the individual an effective amount of a(dimeric) polypeptide or an antibody as reported herein to transport asoluble receptor ligand from the eye over the blood-ocular-barrier intothe blood circulation.

In one embodiment the (dimeric) polypeptide is a bispecific antibody. Inone embodiment the bispecific antibody is a bivalent bispecificantibody. In one embodiment the bispecific antibody is a tetravalentbispecific antibody.

In one embodiment the (dimeric) polypeptide is a trispecific antibody.In one embodiment the trispecific antibody is a trivalent trispecificantibody. In one embodiment the trispecific antibody is a tetravalenttrispecific antibody.

In one embodiment the first polypeptide further comprises the mutationsY349C, T366S, L368A and Y407V, and the second polypeptide furthercomprises the mutations S354C and T366W.

In one embodiment the first polypeptide further comprises the mutationsS354C, T366S, L368A and Y407V and the second polypeptide furthercomprises the mutations Y349C and T366W.

In one embodiment

-   -   i) the first and the second polypeptide comprise the mutations        H310A, H433A and Y436A, or    -   ii) the first and the second polypeptide further comprise the        mutations L251D, L314D and L432D, or    -   iii) the first and the second polypeptide further comprise the        mutations L251S, L314S and L432S, or    -   iv) the first polypeptide comprises the mutations I253A, H310A        and H435A and the second polypeptide comprises the mutations        H310A, H433A and Y436A, or    -   v) the first polypeptide further comprises the mutations I253A,        H310A and H435A and the second polypeptide further comprises the        mutations L251D, L314D and L432D, or    -   vi) the first polypeptide further comprises the mutations I253A,        H310A and H435A and the second polypeptide further comprises the        mutations L251S, L314S and L432S.

In one embodiment the (dimeric) polypeptide is a CrossMab.

In one embodiment the (dimeric) polypeptide is an Fc-region fusionpolypeptide.

In one embodiment the antibody or the Fc-region fusion polypeptide is ofthe subclass IgG1. In one embodiment the antibody or the Fc-regionfusion polypeptide further comprise the mutations L234A and L235A. Inone embodiment the antibody or the Fc-region fusion polypeptide furthercomprise the mutation P329G.

In one embodiment the antibody or the Fc-region fusion polypeptide is ofthe subclass IgG2. In one embodiment the antibody or the Fc-regionfusion polypeptide further comprise the mutations V234A, G237A, P238S,H268A, V309L, A330S, and P331S.

In one embodiment the antibody or the Fc-region fusion polypeptide is ofthe subclass IgG4. In one embodiment the antibody or the Fc-regionfusion polypeptide further comprise the mutations S228P and L235E. Inone embodiment the antibody or the Fc-region fusion polypeptide furthercomprise the mutation P329G.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Scheme of concept and advantages of anti-VEGF/ANG2 antibodiesof the IgG1 or IgG4 subclass with IHH-AAA mutation (combination ofmutations I253A, H310A and H435A (numbering according to the Kabat EUindex numbering system)).

FIG. 2 : Small-scale DLS-based viscosity measurement:

Extrapolated viscosity at 150 mg/mL in 200 mM arginine/succinate buffer,pH 5.5 (comparison of anti-VEGF/ANG2 antibody VEGF/ANG2-0016 (withIHH-AAA mutation) with reference antibody VEGF/ANG2-0015 (without suchIHH-AAA mutation)).

FIG. 3 : DLS Aggregation depending on temperature (including DLSaggregation onset temperature) in 20 mM histidine buffer, 140 mM NaCl,pH 6.0 (comparison of anti-VEGF/ANG2 antibody as reported hereinVEGF/ANG2-0016 (with IHH-AAA mutation) with reference antibodyVEGF/ANG2-0015 (without such IHH-AAA mutation)).

FIG. 4 : Seven day storage at 40° C. at 100 mg/mL (decrease of Main Peakand High Molecular Weight (HMW) increase) (comparison of anti-VEGF/ANG2antibody as reported herein VEGF/ANG2-0016 (with IHH-AAA mutation) whichshowed a lower aggregation with reference antibody VEGF/ANG2-0015(without such IHH-AAA mutation)).

FIGS. 5A and 5B: FcRn steady state affinity of 5A: VEGF/ANG2-0015(without IHH-AAA mutation) and 5B: VEGF/ANG2-0016 (with IHH-AAAmutation).

FIG. 6 : FcgammaRIIIa interaction measurement of VEGF/ANG2-0015 withoutIHH-AAA mutation and VEGF/ANG2-0016 with IHH-AAA mutation (both are IgG1subclass with P329G LALA mutations; as controls an anti-digoxygeninantibody (anti-Dig antibody) of IgG1 subclass and an IgG4 based antibodywere used).

FIG. 7A: Schematic pharmacokinetic (PK) ELISA assay principle fordetermination of concentrations of anti-VEGF/ANG2 antibodies in serumand whole eye lysates.

FIG. 7B: Serum concentration after intravenous (i.v.) application:comparison of VEGF/ANG2-0015 without IHH-AAA mutation and VEGF/ANG2-0016with IHH-AAA mutation.

FIG. 7C: Serum concentration after intravitreal application: comparisonof VEGF/ANG2-0015 without IHH-AAA mutation and VEGF/ANG2-0016 withIHH-AAA mutation.

FIG. 7D: Eye lysates concentration of VEGF/ANG2-0016 (with IHH-AAAmutation) in right and left eye (after intravitreal application onlyinto the right eye in comparison to intravenous application):significant concentrations could be detected only in the right eye afterintravitreal application; after intravenous application no concentrationin eye lysates could be detected due to the low serum half-life ofVEGF/ANG2-0016 (with IHH-AAA mutation).

FIG. 7E: Eye lysates concentration of VEGF/ANG2-0015 (without IHH-AAAmutation) in right and left eye (after intravitreal application onlyinto the right eye in comparison to intravenous application): in theright eye (and to some extent in the left eye) after intravitrealapplication concentrations of VEGF/ANG2-0015 could be detected; thisindicates the diffusion from the right eye into serum and from thereinto the left eye, which can be explained by the long half-life ofVEGF/ANG2-0015 (without IHH-AAA mutation); after intravenous applicationalso significant concentrations in eye lysates of both eyes could bedetected due to diffusion into the eyes of the serum-stableVEGF/ANG2-0015 (without IHH-AAA mutation).

FIGS. 8A-8C: Antibodies engineered with respect to their ability to bindFcRn display prolonged (YTE mutation) or shortened (IHH-AAA mutation) invivo half-lives, enhanced (YTE mutation) or reduced binding (IHH-AAAmutation) compared to the reference wild-type (wt) antibody in SPRanalysis as well as enhanced or reduced retention time in FcRn columnchromatography; FIG. 8A) PK data after single i.v. bolus application of10 mg/kg into huFcRn transgenic male C57BL/6J mice+/−276: AUC data forwt IgG as well as YTE and IHH-AAA Fc-region-modified IgGs; FIG. 8B)BIAcore sensorgram; FIG. 8C) FcRn affinity column elution; wild-typeanti-IGF-1R antibody (reference), YTE-mutant of anti-IGF-1R antibody,IHH-AAA-mutant of anti-IGF-1R antibody.

FIG. 9 : Change of retention time in an FcRn affinity chromatographydepending on the number of mutations introduced into the Fc-region.

FIG. 10 : Change of FcRn-binding depending on asymmetric distribution ofmutations introduced into the Fc-region.

FIG. 11 : Elution chromatogram of a bispecific anti-VEGF/ANG2 antibody(VEGF/ANG2-0121) with the combination of the mutations H310A, H433A andY436A in both heavy chains from two consecutive protein A affinitychromatography columns.

FIG. 12 : Elution chromatogram of an anti-IGF-1R antibody (IGF-1R-0045)with the mutations H310A, H433A and Y436A in both heavy chains from aprotein A affinity chromatography column.

FIG. 13 : Binding of IgG Fc-region modified anti-VEGF/ANG2 antibodies toimmobilized protein A on a CM5 chip.

FIG. 14 : Elution chromatogram of different anti-VEGF/ANG2 antibodies onan FcRn affinity column.

FIG. 15 : Binding of different fusion polypeptides to Staphylococcalprotein A (SPR).

FIG. 16 : Binding of different anti-VEGF/ANG2 antibody and anti-IGF-1Rantibody mutants to immobilized protein A (SPR).

FIG. 17 : Comparison of serum concentrations after intravenousapplication of antibodies IGF-1R 0033, 0035 and 0045.

FIG. 18 : Comparison of eye lysate concentration after intravitreal andintravenous application of antibody IGF-1R 0033.

FIG. 19 : Comparison of eye lysate concentration after intravitreal andintravenous application of antibody IGF-1R 0035.

FIG. 20 : Comparison of eye lysate concentration after intravitreal andintravenous application of antibody IGF-1R 0045.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

The term “about” denotes a range of +/−20% of the thereafter followingnumerical value. In one embodiment the term “about” denotes a range of+/−10% of the thereafter following numerical value. In one embodimentthe term “about” denotes a range of +/−5% of the thereafter followingnumerical value.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencealterations. In some embodiments, the number of amino acid alterationsare 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4or less, 3 or less, or 2 or less. In some embodiments, the VL acceptorhuman framework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “alteration” denotes the mutation (substitution), insertion(addition), or deletion of one or more amino acid residues in a parentantibody or fusion polypeptide, e.g. a fusion polypeptide comprising atleast an FcRn binding portion of an Fc-region, to obtain a modifiedantibody or fusion polypeptide. The term “mutation” denotes that thespecified amino acid residue is substituted for a different amino acidresidue. For example, the mutation L234A denotes that the amino acidresidue lysine at position 234 in an antibody Fc-region (polypeptide) issubstituted by the amino acid residue alanine (substitution of lysinewith alanine) (numbering according to the Kabat EU index numberingsystem).

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and is referred to as“numbering according to Kabat” herein. Specifically the Kabat numberingsystem (see pages 647-660) of Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) is used for the light chainconstant domain CL of kappa and lambda isotype and the Kabat EU indexnumbering system (see pages 661-723) is used for the constant heavychain domains (CH1, Hinge, CH2 and CH3).

A “naturally occurring amino acid residue” denotes an amino acid residuefrom the group consisting of alanine (three letter code: Ala, one lettercode: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp,D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E),glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu,L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F),proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophane (Trp,W), tyrosine (Tyr, Y), and valine (Val, V).

The term “amino acid mutation” denotes the substitution of at least oneexisting amino acid residue with another different amino acid residue(=replacing amino acid residue). The replacing amino acid residue may bea “naturally occurring amino acid residues” and selected from the groupconsisting of alanine (three letter code: ala, one letter code: A),arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G),histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys,K), methionine (met, M), phenylalanine (phe, F), proline (pro, P),serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr,Y), and valine (val, V). The replacing amino acid residue may be a“non-naturally occurring amino acid residue.” See e.g. U.S. Pat. No.6,586,207, WO 98/48032, WO 03/073238, US 2004/0214988, WO 2005/35727, WO2005/74524, Chin, J. W., et al., J. Am. Chem. Soc. 124 (2002) 9026-9027;Chin, J. W. and Schultz, P. G., ChemBioChem 11 (2002) 1135-1137; Chin,J. W., et al., Proc Natl Acad Sci USA (2002) 99(17): 11020-11024; and,Wang, L. and Schultz, P. G., Chem. (2002) 1-10 (all entirelyincorporated by reference herein).

The term “amino acid insertion” denotes the (additional) incorporationof at least one amino acid residue at a predetermined position in anamino acid sequence. In one embodiment the insertion will be theinsertion of one or two amino acid residues. The inserted amino acidresidue(s) can be any naturally occurring or non-naturally occurringamino acid residue.

The term “amino acid deletion” denotes the removal of at least one aminoacid residue at a predetermined position in an amino acid sequence.

The term “ANG-2” as used herein refers to human angiopoietin-2 (ANG-2)(alternatively abbreviated with ANGPT2 or ANG2) (SEQ ID NO: 31) which isdescribed e.g. in Maisonpierre, P. C., et al, Science 277 (1997) 55-60and Cheung, A. H., et al., Genomics 48 (1998) 389-91. Theangiopoietins-1 (SEQ ID NO: 32) and -2 were discovered as ligands forthe Ties, a family of tyrosine kinases that is selectively expressedwithin the vascular endothelium (Yancopoulos, G. D., et al., Nature 407(2000) 242-248). There are now four definitive members of theangiopoietin family. Angiopoietin-3 and -4 (ANG-3 and ANG-4) mayrepresent widely diverged counterparts of the same gene locus in mouseand man (Kim, I., et al., FEBS Let, 443 (1999) 353-356; Kim, I., et al.,J. Biol. Chem. 274 (1999) 26523-26528). ANG-1 and ANG-2 were originallyidentified in tissue culture experiments as agonist and antagonist,respectively (see for ANG-1: Davis, S., et al., Cell 87 (1996)1161-1169; and for ANG-2: Maisonpierre, P. C., et al., Science 277(1997) 55-60). All of the known angiopoietins bind primarily to Tie2(SEQ ID NO: 33), and both ANG-1 and -2 bind to Tie2 with an affinity of3 nM (Kd) (Maisonpierre, P. C., et al., Science 277 (1997) 55-60).

The term “antibody” is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to, monoclonalantibodies, multispecific antibodies (e.g. bispecific antibodies,trispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-, and/or protein A and/or FcRn-binding activity.

The term “asymmetric Fc-region” denotes a pair of Fc-region polypeptidesthat have different amino acid residues at corresponding positionsaccording to the Kabat EU index numbering system.

The term “asymmetric Fc-region with respect to FcRn binding” denotes anFc-region that consists of two polypeptide chains that have differentamino acid residues at corresponding positions, whereby the positionsare determined according to the Kabat EU index numbering system, wherebythe different positions affect the binding of the Fc-region to the humanneonatal Fc-receptor (FcRn). For the purpose herein, the differencesbetween the two polypeptide chains of the Fc-region in an “asymmetricFc-region with respect to FcRn binding” do not include differences thathave been introduced to facilitate the formation of heterodimericFc-regions, e.g. for the production of bispecific antibodies. Thesedifferences can also be asymmetric, i.e. the two chains have differencesat non-corresponding amino acid residues according to the Kabat EU indexnumbering system. These differences facilitate heterodimerization andreduce homodimerization. Examples of such differences are the so-called“knobs into holes” substitutions (see, e.g., U.S. Pat. No. 7,695,936 andUS 2003/0078385). The following knobs and holes substitutions in theindividual polypeptide chains of an Fc-region of an IgG antibody ofsubclass IgG1 have been found to increase heterodimer formation: 1)Y407T in one chain and T366Y in the other chain; 2) Y407A in one chainand T366W in the other chain; 3) F405A in one chain and T394W in theother chain; 4) F405W in one chain and T394S in the other chain; 5)Y407T in one chain and T366Y in the other chain; 6) T366Y and F405A inone chain and T394W and Y407T in the other chain; 7) T366W and F405W inone chain and T394S and Y407A in the other chain; 8) F405W and Y407A inone chain and T366W and T394S in the other chain; and 9) T366W in onechain and T366S, L368A, and Y407V in the other chain, whereby the lastlisted is especially suited. In addition, changes creating new disulfidebridges between the two Fc-region polypeptide chains facilitateheterodimer formation (see, e.g., US 2003/0078385). The followingsubstitutions resulting in appropriately spaced apart cysteine residuesfor the formation of new intra-chain disulfide bonds in the individualpolypeptide chains of an Fc-region of an IgG antibody of subclass IgG1have been found to increase heterodimer formation: Y349C in one chainand S354C in the other; Y349C in one chain and E356C in the other; Y349Cin one chain and E357C in the other; L351C in one chain and S354C in theother; T394C in one chain and E397C in the other; or D399C in one chainand K392C in the other. Further examples of heterodimerizationfacilitating amino acid changes are the so-called “charge pairsubstitutions” (see, e.g., WO 2009/089004). The following charge pairsubstitutions in the individual polypeptide chains of an Fc-region of anIgG antibody of subclass IgG1 have been found to increase heterodimerformation: 1) K409D or K409E in one chain and D399K or D399R in theother chain; 2) K392D or K392E in one chain and D399K or D399R in theother chain; 3) K439D or K439E in one chain and E356K or E356R in theother chain; 4) K370D or K370E in one chain and E357K or E357R in theother chain; 5) K409D and K360D in one chain plus D399K and E356K in theother chain; 6) K409D and K370D in one chain plus D399K and E357K in theother chain; 7) K409D and K392D in one chain plus D399K, E356K, andE357K in the other chain; 8) K409D and K392D in one chain and D399K inthe other chain; 9) K409D and K392D in one chain and D399K and E356K inthe other chain; 10) K409D and K392D in one chain and D399K and D357K inthe other chain; 11) K409D and K370D in one chain and D399K and D357K inthe other chain; 12) D399K in one chain and K409D and K360D in the otherchain; and 13) K409D and K439D in one chain and D399K and E356K on theother.

The term “binding (to an antigen)” denotes the binding of an antibody toits antigen in an in vitro assay, in one embodiment in a binding assayin which the antibody is bound to a surface and binding of the antigento the antibody is measured by Surface Plasmon Resonance (SPR). Bindingmeans a binding affinity (K_(D)) of 10⁻⁸ M or less, in some embodimentsof 10⁻¹³ to 10⁻⁸M, in some embodiments of 10⁻¹³ to 10⁻⁹M.

Binding can be investigated by a BIAcore assay (GE Healthcare BiosensorAB, Uppsala, Sweden). The affinity of the binding is defined by theterms k_(a) (rate constant for the association of the antibody from theantibody/antigen complex), k_(d) (dissociation constant), andK_(D)(k_(d)/k_(a)).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The term “CH2-domain” denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 231 to EUposition 340 (EU numbering system according to Kabat). In one embodimenta CH2 domain has the amino acid sequence of SEQ ID NO: 09: APELLGGPSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQ ESTYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAK.

The term ““CH3-domain” denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 341 to EUposition 446. In one embodiment the CH3 domain has the amino acidsequence of SEQ ID NO: 10: GQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYTQKSLSLSPG.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “comparable length” denotes that two polypeptides comprise theidentical number of amino acid residues or can be different in length byone or more and up to 10 amino acid residues at most. In one embodimentthe (Fc-region) polypeptides comprise the identical number of amino acidresidues or differ by a number of from 1 to 10 amino acid residues. Inone embodiment the (Fc-region) polypeptides comprise the identicalnumber of amino acid residues or differ by a number of from 1 to 5 aminoacid residues. In one embodiment the (Fc-region) polypeptides comprisethe identical number of amino acid residues or differ by a number offrom 1 to 3 amino acid residues.

“Effector functions” refer to those biological activities attributableto the Fc-region of an antibody, which vary with the antibody class.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B-cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc-fusion polypeptide” denotes a fusion of a binding domain(e.g. an antigen binding domain such as a single chain antibody, or apolypeptide such as a ligand of a receptor) with an antibody Fc-regionthat exhibits the desired target-, protein A- and FcRn-binding activity.

The term “Fc-region of human origin” denotes the C-terminal region of animmunoglobulin heavy chain of human origin that contains at least a partof the hinge region, the CH2 domain and the CH3 domain. In oneembodiment, a human IgG heavy chain Fc-region extends from Cys226, orfrom Pro230, to the carboxyl-terminus of the heavy chain. In oneembodiment the Fc-region has the amino acid sequence of SEQ ID NO: 60.However, the C-terminal lysine (Lys447) of the Fc-region may or may notbe present.

The term “FcRn” denotes the human neonatal Fc-receptor. FcRn functionsto salvage IgG from the lysosomal degradation pathway, resulting inreduced clearance and increased half-life. The FcRn is a heterodimericprotein consisting of two polypeptides: a 50 kDa class I majorhistocompatibility complex-like protein (α-FcRn) and a 15 kDaβ2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3portion of the Fc-region of IgG. The interaction between IgG and FcRn isstrictly pH dependent and occurs in a 1:2 stoichiometry, with one IgGbinding to two FcRn molecules via its two heavy chains (Huber, A. H., etal., J. Mol. Biol. 230 (1993) 1077-1083). FcRn binding occurs in theendosome at acidic pH (pH<6.5) and IgG is released at the neutral cellsurface (pH of about 7.4). The pH-sensitive nature of the interactionfacilitates the FcRn-mediated protection of IgGs pinocytosed into cellsfrom intracellular degradation by binding to the receptor within theacidic environment of endosomes. FcRn then facilitates the recycling ofIgG to the cell surface and subsequent release into the blood streamupon exposure of the FcRn-IgG complex to the neutral pH environmentoutside the cell.

The term “FcRn binding portion of an Fc-region” denotes the part of anantibody heavy chain polypeptide that extends approximately from EUposition 243 to EU position 261 and approximately from EU position 275to EU position 293 and approximately from EU position 302 to EU position319 and approximately from EU position 336 to EU position 348 andapproximately from EU position 367 to EU position 393 and EU position408 and approximately from EU position 424 to EU position 440. In oneembodiment one or more of the following amino acid residues according tothe EU numbering of Kabat are altered F243, P244, P245 P, K246, P247,K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259,T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285,N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305,L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317,E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345,P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378,V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391,T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433,N434, H435, Y436, T437, Q438, K439, and S440 (EU numbering).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “full length antibody” denotes an antibody having a structuresubstantially similar to a native antibody structure comprising fourpolypeptides or having heavy chains that contain an Fc-region as definedherein. A full length antibody may comprise further domains, such ase.g. a scFv or a scFab conjugated to one or more of the chains of thefull length antibody. These conjugates are also encompassed by the termfull length antibody.

The term “dimeric polypeptide” denotes a complex comprising at least twopolypeptides that are associated covalently. The complex may comprisefurther polypeptides that are also associated covalently ornon-covalently with the other polypeptides. In one embodiment thedimeric polypeptide comprises two or four polypeptides.

The terms “heterodimer” or “heterodimeric” denote a molecule thatcomprises two polypeptides (e.g. of comparable length), wherein the twopolypeptides have an amino acid sequence that have at least onedifferent amino acid residue in a corresponding position, wherebycorresponding position is determined according to the Kabat EU indexnumbering system.

The terms “homodimer” and “homodimeric” denote a molecule that comprisestwo polypeptides of comparable length, wherein the two polypeptides havean amino acid sequence that is identical in corresponding positions,whereby corresponding positions are determined according to the Kabat EUindex numbering system.

A dimeric polypeptide as reported herein can be homodimeric orheterodimeric which is determined with respect to mutations orproperties in focus. For example, with respect to FcRn and/or protein Abinding (i.e. the focused on properties) a dimeric polypeptide ishomodimeric (i.e. both polypeptides of the dimeric polypeptide comprisethese mutations) with respect to the mutations H310A, H433A and Y436A(these mutations are in focus with respect to FcRn and/or protein Abinding property of the dimeric polypeptide) but at the same timeheterodimeric with respect to the mutations Y349C, T366S, L368A andY407V (these mutations are not in focus as these mutations are directedto the heterodimerization of the dimeric polypeptide and not to theFcRn/protein A binding properties) as well as the mutations S354C andT366W, respectively (the first set is comprised only in the firstpolypeptide whereas the second set is comprised only in the secondpolypeptide). Further for example, a dimeric polypeptide as reportedherein can be heterodimeric with respect to the mutations I253A, H310A,H433A, H435A and Y436A (i.e. these mutations are directed all to theFcRn and/or protein A binding properties of the dimeric polypeptide),i.e. one polypeptide comprises the mutations I253A, H310A and H435A,whereas the other polypeptide comprises the mutations H310A, H433A andY436A.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Bethesda Md. (1991), NIH Publication 91-3242, Vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

The term “derived from” denotes that an amino acid sequence is derivedfrom a parent amino acid sequence by introducing alterations at at leastone position. Thus, a derived amino acid sequence differs from thecorresponding parent amino acid sequence at at least one correspondingposition (numbering according to Kabat EU index for antibodyFc-regions). In one embodiment an amino acid sequence derived from aparent amino acid sequence differs by one to fifteen amino acid residuesat corresponding positions. In one embodiment an amino acid sequencederived from a parent amino acid sequence differs by one to ten aminoacid residues at corresponding positions. In one embodiment an aminoacid sequence derived from a parent amino acid sequence differs by oneto six amino acid residues at corresponding positions. Likewise, aderived amino acid sequence has a high amino acid sequence identity toits parent amino acid sequence. In one embodiment an amino acid sequencederived from a parent amino acid sequence has 80% or more amino acidsequence identity. In one embodiment an amino acid sequence derived froma parent amino acid sequence has 90% or more amino acid sequenceidentity. In one embodiment an amino acid sequence derived from a parentamino acid sequence has 95% or more amino acid sequence identity.

The term “human Fc-region polypeptide” denotes an amino acid sequencewhich is identical to a “native” or “wild-type” human Fc-regionpolypeptide. The term “variant (human) Fc-region polypeptide” denotes anamino acid sequence which derived from a “native” or “wild-type” humanFc-region polypeptide by virtue of at least one “amino acid alteration.”A “human Fc-region” consists of two human Fc-region polypeptides. A“variant (human) Fc-region” consists of two Fc-region polypeptides,whereby both can be variant (human) Fc-region polypeptides, or one is ahuman Fc-region polypeptide and the other is a variant (human) Fc-regionpolypeptide.

In one embodiment the human Fc-region polypeptide has the amino acidsequence of a human IgG1 Fc-region polypeptide of SEQ ID NO: 60, or of ahuman IgG2 Fc-region polypeptide of SEQ ID NO: 61, or of a human IgG4Fc-region polypeptide of SEQ ID NO: 63 with the mutations as reportedherein. In one embodiment the variant (human) Fc-region polypeptide isderived from an Fc-region polypeptide of SEQ ID NO: 60, or 61, or 63 andhas at least one amino acid mutation compared to the Fc-regionpolypeptide of SEQ ID NO: 60, or 61, or 63. In one embodiment thevariant (human) Fc-region polypeptide comprises/has from about one toabout ten amino acid mutations, and in one embodiment from about one toabout five amino acid mutations. In one embodiment the variant (human)Fc-region polypeptide has at least about 80% homology with a humanFc-region polypeptide of SEQ ID NO: 60, or 61, or 63. In one embodimentthe variant (human) Fc-region polypeptide has least about 90% homologywith a human Fc-region polypeptide of SEQ ID NO: 60, or 61, or 63. Inone embodiment the variant (human) Fc-region polypeptide has at leastabout 95% homology with a human Fc-region polypeptide of SEQ ID NO: 60,or 61, or 63.

The variant (human) Fc-region polypeptide derived from a human Fc-regionpolypeptide of SEQ ID NO: 60, or 61, or 63 is defined by the amino acidalterations that are contained. Thus, for example, the term P329Gdenotes a variant (human) Fc-region polypeptide derived human Fc-regionpolypeptide with the mutation of proline to glycine at amino acidposition 329 relative to the human Fc-region polypeptide of SEQ ID NO:60, or 61, or 63.

For all positions discussed in the present invention, numbering isaccording to the Kabat EU index numbering system.

A human IgG1 Fc-region polypeptide has the following amino acidsequence:

(SEQ ID NO: 60) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with the mutationsL234A, L235A has the following amino acid sequence:

(SEQ ID NO: 64) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with Y349C, T366S,L368A and Y407V mutations has the following amino acid sequence:

(SEQ ID NO: 65) DKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with S354C, T366Wmutations has the following amino acid sequence:

(SEQ ID NO: 66) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235Amutations and Y349C, T366S, L368A, Y407V mutations has the followingamino acid sequence:

(SEQ ID NO: 67) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a L234A, L235Aand S354C, T366W mutations has the following amino acid sequence:

(SEQ ID NO: 68) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a P329Gmutation has the following amino acid sequence:

(SEQ ID NO: 69) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235Amutations and P329G mutation has the following amino acid sequence:

(SEQ ID NO: 70) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a P239Gmutation and Y349C, T366S, L368A, Y407V mutations has the followingamino acid sequence:

(SEQ ID NO: 71) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with a P329Gmutation and S354C, T366W mutation has the following amino acidsequence:

(SEQ ID NO: 72) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A,P329G and Y349C, T366S, L368A, Y407V mutations has the following aminoacid sequence:

(SEQ ID NO: 73) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK.

A human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A,P329G mutations and S354C, T366W mutations has the following amino acidsequence:

(SEQ ID NO: 74) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK.

A human IgG4 Fc-region polypeptide has the following amino acidsequence:

(SEQ ID NO: 63) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with S228P andL235E mutations has the following amino acid sequence:

(SEQ ID NO: 75) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with S228P, L235Emutations and P329G mutation has the following amino acid sequence:

(SEQ ID NO: 76) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with S354C, T366Wmutations has the following amino acid sequence:

(SEQ ID NO: 77) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with Y349C, T366S,L368A, Y407V mutations has the following amino acid sequence:

(SEQ ID NO: 78) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235Eand S354C, T366W mutations has the following amino acid sequence:

(SEQ ID NO: 79) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235Eand Y349C, T366S, L368A, Y407V mutations has the following amino acidsequence:

(SEQ ID NO: 80) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a P329Gmutation has the following amino acid sequence:

(SEQ ID NO: 81) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a P239G andY349C, T366S, L368A, Y407V mutations has the following amino acidsequence:

(SEQ ID NO: 82) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a P329G andS354C, T366W mutations has the following amino acid sequence:

(SEQ ID NO: 83) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P,L235E, P329G and Y349C, T366S, L368A, Y407V mutations has the followingamino acid sequence:

(SEQ ID NO: 84) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A human IgG4 Fc-region derived Fc-region polypeptide with a S228P,L235E, P329G and S354C, T366W mutations has the following amino acidsequence:

(SEQ ID NO: 85) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

An alignment of the different human Fc-regions is shown below (Kabat EUindex numbering system):

                                                 2                                                 1                                                 6(IgG1,2,4) IGG1.......... .......... .......... .......... .....EPKSC IGG2.......... .......... .......... .......... .....ERKCC IGG3KTPLGDTTHT CPRCPEPKSC DTPPPCPRCP EPKSCDTPPP CPRCPEPKSC IGG4.......... .......... .......... .......... .....ESKYG-- HINGE ---------------------------------------------         2                     2          3                     5         0                     0 IGG1DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED IGG2...VECPPCP APP.VAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED IGG3DTPPPCPRCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED IGG4...PPCPSCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED-- HINGE -|-- CH2 ------------------------------------                               3                                0                               0 IGG1PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK IGG2PEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK IGG3PEVQFKWYVD GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK IGG4PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK-- CH2 -----------------------------------------------                               3                                5                               0 IGG1CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK IGG2CKVSNKGLPA PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK IGG3CKVSNKALPA PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK IGG4CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK-- CH2 ------- CH2 --|-- CH3 -------------------------                               4                                0                               0 IGG1GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG IGG2GFYPSDISVE WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG IGG3GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG IGG4GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG-- CH3 -----------------------------------------------                            4                             4                            7 IGG1 NVFSCSVMHE ALHNHYTQKS LSLSPGK IGG2NVFSCSVMHE ALHNHYTQKS LSLSPGK IGG3 NIFSCSVMHE ALHNRFTQKS LSLSPGK IGG4NVFSCSVMHE ALHNHYTQKS LSLSLGK -- CH3 ----------------------|

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., the CDRs)correspond to those of a non-human antibody, and all or substantiallyall of the FRs correspond to those of a human antibody. A humanizedantibody optionally may comprise at least a portion of an antibodyconstant region derived from a human antibody. A “humanized form” of anantibody, e.g., a non-human antibody, refers to an antibody that hasundergone humanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and formstructurally defined loops (“hypervariable loops”), and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). HVRs as denoted herein include

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987)        901-917);    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat, E. A. et al., Sequences of Proteins of Immunological        Interest, 5th ed. Public Health Service, National Institutes of        Health, Bethesda, Md. (1991), NIH Publication 91-3242.);    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according to theKabat EU index numbering system (Kabat et al., supra).

The term “IGF-1R” as used herein, refers to any native IGF-1R from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed IGF-1R as well as any form ofIGF-1R that results from processing in the cell. The term alsoencompasses naturally occurring variants of IGF-1R, e.g., splicevariants or allelic variants. The amino acid sequence of human IGF-1R isshown in SEQ ID NO: 11.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., size exclusionchromatography, ion exchange or reverse phase HPLC) methods. For reviewof methods for assessment of antibody purity, see, e.g., Flatman, S. etal., J. Chrom. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-IGF-1R antibody” refers to oneor more nucleic acid molecules encoding antibody heavy and light chains(or fragments thereof), including such nucleic acid molecule(s) in asingle vector or separate vectors, and such nucleic acid molecule(s)present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “peptidic linker” as used herein denotes a peptide with aminoacid sequences, which is in one embodiment of synthetic origin. Thepeptidic linker is in one embodiment a peptide with an amino acidsequence with a length of at least 30 amino acids, in one embodimentwith a length of 32 to 50 amino acids. In one embodiment the peptidiclinker is a peptide with an amino acid sequence with a length of 32 to40 amino acids. In one embodiment the peptidic linker is (GxS)n withG=glycine, S=serine, (x=3, n=8, 9 or 10) or (x=4 and n=6, 7 or 8), inone embodiment with x=4, n=6 or 7, in one embodiment with x=4, n=7. Inone embodiment the peptidic linker is (G₄S)₆G₂.

The term “recombinant antibody,” as used herein, denotes all antibodies(chimeric, humanized and human) that are prepared, expressed, created orisolated by recombinant means. This includes antibodies isolated from ahost cell such as a NS0 or CHO cell, or from an animal (e.g. a mouse)that is transgenic for human immunoglobulin genes, or antibodiesexpressed using a recombinant expression vector transfected into a hostcell. Such recombinant antibodies have variable and constant regions ina rearranged form. The recombinant antibodies can be subjected to invivo somatic hypermutation. Thus, the amino acid sequences of the VH andVL regions of the recombinant antibodies are sequences that, whilederived from and related to human germ line VH and VL sequences, may notnaturally exist within the human antibody germ line repertoire in vivo.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies orFc-region fusion polypeptides as reported herein are used to delaydevelopment of a disease or to slow the progression of a disease.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in a (antibody)molecule. As such, the terms “bivalent,” “tetravalent,” and “hexavalent”denote the presence of two binding site, four binding sites, and sixbinding sites, respectively, in a (antibody) molecule. The bispecificantibodies as reported herein are in one preferred embodiment“bivalent.”

The term “variable region” or “variable domain” refer to the domain ofan antibody heavy or light chain that is involved in binding of theantibody to its antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of an antibody generally havesimilar structures, with each domain comprising four framework regions(FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T. J. etal. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page91). A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively (see, e.g., Portolano, S. et al., J.Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991)624-628).

The term “ocular vascular disease” includes, but is not limited tointraocular neovascular syndromes such as diabetic retinopathy, diabeticmacular edema, retinopathy of prematurity, neovascular glaucoma, retinalvein occlusions, central retinal vein occlusions, macular degeneration,age-related macular degeneration, retinitis pigmentosa, retinalangiomatous proliferation, macular telangectasia, ischemic retinopathy,iris neovascularization, intraocular neovascularization, cornealneovascularization, retinal neovascularization, choroidalneovascularization, and retinal degeneration (see e.g. Garner, A.,Vascular diseases, In: Pathobiology of ocular disease, A dynamicapproach, Garner, A., and Klintworth, G. K., (eds.), 2nd edition, MarcelDekker, New York (1994), pp. 1625-1710).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The term “VEGF” as used herein refers to human vascular endothelialgrowth factor (VEGF/VEGF-A) the 165-amino acid human vascularendothelial cell growth factor (amino acid 27-191 of precursor sequenceof human VEGF165: SEQ ID NO: 30; amino acids 1-26 represent the signalpeptide), and related 121, 189, and 206 vascular endothelial cell growthfactor isoforms, as described by Leung, D. W., et al., Science 246(1989) 1306-1309; Houck et al., Mol. Endocrin. 5 (1991) 1806-1814; Keck,P. J., et al., Science 246 (1989) 1309-1312 and Connolly, D. T., et al.,J. Biol. Chem. 264 (1989) 20017-20024; together with the naturallyoccurring allelic and processed forms of those growth factors. VEGF isinvolved in the regulation of normal and abnormal angiogenesis andneovascularization associated with tumors and intraocular disorders(Ferrara, N., et al., Endocrin. Rev. 18 (1997) 4-25; Berkman, R. A., etal., J. Clin. Invest. 91 (1993) 153-159; Brown, L. F., et al., HumanPathol. 26 (1995) 86-91; Brown, L. F., et al., Cancer Res. 53 (1993)4727-4735; Mattern, J., et al., Brit. J. Cancer. 73 (1996) 931-934; andDvorak, H. F., et al., Am. J. Pathol. 146 (1995) 1029-1039). VEGF is ahomodimeric glycoprotein that has been isolated from several sources andincludes several isoforms. VEGF shows highly specific mitogenic activityfor endothelial cells.

The term “with (the) mutation IHH-AAA” as used herein refers to thecombination of the mutations I253A (Ile253Ala), H310A (His310Ala), andH435A (His435Ala) and the term “with (the) mutation HHY-AAA” as usedherein refers to the combination of the mutations H310A (His310Ala),H433A (His433Ala), and Y436A (Tyr436Ala) and the term “with (the)mutation YTE” as used herein refers to the combination of mutationsM252Y (Met252Tyr), S254T (Ser254Thr), and T256E (Thr256Glu) in theconstant heavy chain region of IgG1 or IgG4 subclass, wherein thenumbering is according to the Kabat EU index numbering system.

The term “with (the) mutations P329G LALA” as used herein refers to thecombination of the mutations L234A (Leu235Ala), L235A (Leu234Ala) andP329G (Pro329Gly) in the constant heavy chain region of IgG1 subclass,wherein the numbering is according to the Kabat EU index numberingsystem. The term “with (the) mutation SPLE” as used herein refers to thecombination of the mutations S228P (Ser228Pro) and L235E (Leu235Glu) inthe constant heavy chain region of IgG4 subclass, wherein the numberingis according to the Kabat EU index numbering system. The term “with(the) mutation SPLE and P329G” as used herein refers to the combinationof the mutations S228P (Ser228Pro), L235E (Leu235Glu) and P329G(Pro329Gly) in the constant heavy chain region of IgG4 subclass, whereinthe numbering is according to the Kabat EU index numbering system.

II. Compositions and Methods

In one aspect, the invention is based, in part, on the finding that theintroduction of the mutation Y436A in one or both Fc-region polypeptidesof an Fc-region can increase the binding of an Fc-region toStaphylococcal protein A.

In one aspect, the invention is based, in part, on the finding thatspecific mutations or combination of mutations which influence thebinding of an immunoglobulin Fc-region to the neonatal Fc-receptor(FcRn), i.e. which reduce or even eliminate the binding of the Fc-regionto FcRn, do not simultaneously eliminate the binding of the Fc-region toStaphylococcal protein A. This has a profound effect on the purificationprocess that can be employed as, e.g. no specific and species limitedaffinity chromatography materials, such as e.g. KappaSelect which onlybinds to antibodies comprising a kappa light chain, are required. Thus,with the combination of mutations as reported herein it is possible atthe same time to reduce or even eliminate the binding to FcRn whilemaintaining the binding to Staphylococcal protein A.

In one aspect, the invention is based, in part, on the finding that byusing different mutations in the Fc-regions of each heavy chain of aheterodimeric molecule (such as e.g. a bispecific antibody) aheterodimeric molecule can be provided that on the one hand has areduced or even eliminated binding to FcRn but on the other handmaintains the ability to bind to Staphylococcal protein A. This bindingto Staphylococcal protein A can be used to separate the heterodimericmolecule from homodimeric by-products. For example by combining themutations I253A, H310A and H435A in one heavy chain Fc-region with themutations H310A, H433A and Y436A in the other heavy chain Fc-regionusing the knobs-into-hole approach a heterodimeric Fc-region can beobtained that on the one hand does not bind to FcRn (both sets ofmutations are silent with respect to the human FcRn) but maintainsbinding to Staphylococcal protein A (the heavy chain Fc-region with themutations I253A, H310A and H435A does not bind to FcRn and does not bindto Staphylococcal protein A, whereas the heavy chain Fc-region with themutations H310A, H433A and Y436A does not bind to FcRn but does stillbind to Staphylococcal protein A). Thus, standard protein A affinitychromatography can be used to remove the homodimeric hole-holeby-product as this no longer binds to Staphylococcal protein A). Thus,by combining the knobs-into-holes approach with the mutations I253A,H310A and H435A in the hole chain and the mutations H310A, H433A andY436A in the knobs chain, the purification/separation of theheterodimeric knobs-into-holes product from the homodimeric hole-holeby-product can be facilitated.

In one aspect, the invention is based, in part, on the finding thatantibodies for intravitreal application are beneficial that do not haveFcRn-binding as these antibodies can cross the blood-retinal-barrier, donot have substantially prolonged or shortened half-lives in the eye, andare cleared fast from the blood circulation resulting in no or verylimited systemic side effects outside the eye. Antibodies of theinvention are useful, e.g., for the diagnosis or treatment of ocularvascular diseases.

The invention is based, at least in part, on the finding that by usingdifferent mutations in each of the Fc-region polypeptides of anFc-region in a heterodimeric molecule (such as e.g. a bispecificantibody) a heterodimeric molecule can be provided that has tailor-madeFcRn-binding and therewith antibodies can be provided that have atailor-made systemic half-life.

The combination of mutations I253A, H310A, H435A, or L251D, L314D,L432D, or L251S, L314S, L432S result in a loss of the binding to proteinA, whereas the combination of mutations I253A, H310A, H435A, or H310A,H433A, Y436A, or L251D, L314D, L432D result in a loss of the binding tothe human neonatal Fc receptor.

The following table presents an exemplary overview of the amino acidresidues in an Fc-region that are involved in interactions or have beenchanged to modify interactions.

interaction with KiH protein A effect of mutations on residue protein AFcRn knob hole binding FcRn binding Pro238 P238A increase Thr250T250Q/M428L increase Leu251 main-chain contact Met252 hydrophobic M252Wincrease; packing M252Y increase; M252Y/T256Q increase; M252F/T256Dincrease; M252Y/S254T/T256E increase Ile253 main-chain interaction I253Areduction contact; hydrogen bonding; significant binding reduction ifmutated to Ala Ser254 polar S254A reduction; interaction;M252Y/S254T/T256E hydrogen increase bonding Arg255 salt-bridge R255Areduction Thr256 T256A increase; T256Q increase; T256P increase;M252Y/T256Q reduction; M252F/T256D reduction; M252Y/S254T/T256E increasePro257 P257I/Q311I increase; P257I/N434H increase Glu272 E272A increaseAsp280 D280K increase His285 reduction Lys288 K288A reduction;K288A/N434A increase Val305 V305A increase Thr307 T307A increase;T307A/E380A/N434A increase; T307Q/N434A increase; T307Q/N434S increase;T307Q/E380A/N434A increase Val308 V308P/N434A increase Leu309 L309Areduction His310 interaction H310A reduction; H310Q/H433N reductionGln311 polar or Q311A increase; charged P257I/Q311I increase interactionAsp312 D312A increase Leu314 hydrophobic interaction Lys317 K317Aincrease Ala339 A339T increase Tyr349 Y349C Ser354 S354C Thr366 T366WT366S Leu368 L368A Asp376 D376A increase; D376V/N434H increase Ala378A378Q increase Glu380 salt-bridge E380A increase E380A/N434A increase;T307A/E380A/N434A increase; T307Q/E380A/N434A increase Glu382 E382Aincrease Gly385 G385H increase; G385A/Q386P/N389S increase Gln386G385A/Q386P/N389S increase Asn389 G385A/Q386P/N389S increase Tyr407Y407V Ser415 S415A reduction Ser424 S424A increase Met428 M428Lincrease; T250Q/M428L increase Leu432 polar or charged interactionHis433 polar or interaction H433A reduction; charged H310Q/H433Ninteraction; reduction; salt-bridge H433K/N434F/Y436H increase;H433R/N434Y/Y436H increase; H433K/N434F increase Asn434 hydrogeninteraction N434W/Y/F/A/H bonding; increase; significant K288A/N434Aincrease; binding E380A/N434A increase; reduction if T307A/E380A/N434Areplaced by increase; Ala N434F/Y436H increase; H433K/N434F/Y436Hincrease; H433R/N434Y/Y436H increase; H433K/N434F increase; P257I/N434Hincrease; D376V/N434H increase; T307Q/N434A increase; T307Q/N434Sincrease; V308P/N434A increase; T307Q/E380A/N434A increase His435hydrophobic interaction H435R/Y436F H435A reduction; packing; eliminatesH435R reduction significant binding to binding protein A reduction ifmutated to Ala Tyr436 hydrophobic interaction H435R/Y436F Y436Areduction; packing; eliminates N434F/Y436H increase; significant bindingto H433K/N434F/Y436H binding protein A increase; reduction ifH433R/N434Y/Y436H replaced by increase Ala

The modifications as reported herein, which can be used in combinationwith the mutation Y436A, alter the binding specificity for one or moreFc receptors such as the human FcRn. At the same time, some of themutations which alter the binding to human FcRn also alter the bindingto Staphylococcal protein A. This reduction in binding to Staphylococcalprotein A can be reduced or even overcome by using the additionalmutation Y436A.

In one embodiment the combination of mutations as reported herein doesalter or does substantially alter the serum half-life of the dimericpolypeptide as compared with a corresponding dimeric polypeptide thatlacks this combination of mutations. In one embodiment the combinationof mutations further does not alter or does not substantially alter thebinding of the dimeric polypeptide to Staphylococcal protein A ascompared with a corresponding dimeric polypeptide that lacks thiscombination of mutations.

A. The Neonatal Fc-Receptor (FcRn)

The neonatal Fc-receptor (FcRn) is important for the metabolic fate ofantibodies of the IgG class in vivo. The FcRn functions to salvagewild-type IgG from the lysosomal degradation pathway, resulting inreduced clearance and increased half-life. It is a heterodimeric proteinconsisting of two polypeptides: a 50 kDa class I majorhistocompatibility complex-like protein (α-FcRn) and a 15 kDaβ2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3portion of the Fc-region of an antibody of the class IgG. Theinteraction between an antibody of the class IgG and the FcRn is pHdependent and occurs in a 1:2 stoichiometry, i.e. one IgG antibodymolecule can interact with two FcRn molecules via its two heavy chainFc-region polypeptides (see e.g. Huber, A. H., et al., J. Mol. Biol. 230(1993) 1077-1083).

Thus, the in vitro FcRn binding properties/characteristics of an IgG areindicative of its in vivo pharmacokinetic properties in the bloodcirculation.

In the interaction between the FcRn and the Fc-region of an antibody ofthe IgG class, different amino acid residues of the heavy chain CH2- andCH3-domain participate. The amino acid residues interacting with theFcRn are located approximately between EU position 243 and EU position261, approximately between EU position 275 and EU position 293,approximately between EU position 302 and EU position 319, approximatelybetween EU position 336 and EU position 348, approximately between EUposition 367 and EU position 393, at EU position 408, and approximatelybetween EU position 424 and EU position 440. More specifically, thefollowing amino acid residues according to the EU numbering of Kabat areinvolved in the interaction between the Fc-region and the FcRn: F243,P244, P245 P, K246, P247, K248, D249, T250, L251, M252, I253, S254,R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279,D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292,E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312,W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340,G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373,P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N384, G385,Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428,H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439, andS440.

Site-directed mutagenesis studies have proven that the critical bindingsites in the Fc-region of IgGs for FcRn are Histidine 310, Histidine435, and Isoleucine 253 and to a lesser extent Histidine 433 andTyrosine 436 (see e.g. Kim, J. K., et al., Eur. J. Immunol. 29 (1999)2819-2825; Raghavan, M., et al., Biochem. 34 (1995) 14649-14657;Medesan, C., et al., J Immunol. 158 (1997) 2211-2217).

Methods to increase IgG binding to FcRn have been performed by mutatingIgG at various amino acid residues: Threonine 250, Methionine 252,Serine 254, Threonine 256, Threonine 307, Glutamic acid 380, Methionine428, Histidine 433, and Asparagine 434 (see Kuo, T. T., et al., J. Clin.Immunol. 30 (2010) 777-789).

In some cases, antibodies with reduced half-life in the bloodcirculation are desired. For example, drugs for intravitreal applicationshould have a long half-live in the eye and a short half-life in theblood circulation of the patient. Such antibodies also have theadvantage of increased exposure to a disease site, e.g. in the eye.

Different mutations that influence the FcRn binding and therewith thehalf-life in the blood circulation are known. Fc-region residuescritical to the mouse Fc-region-mouse FcRn interaction have beenidentified by site-directed mutagenesis (see e.g. Dall'Acqua, W. F., etal. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434,and H435 (EU numbering according to Kabat) are involved in theinteraction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533-2536;Firan, M., et al., Int. Immunol. 13 (2001) 993-1002; Kim, J. K., et al.,Eur. J. Immunol. 24 (1994) 542-548). Residues I253, H310, and H435 werefound to be critical for the interaction of human Fc with murine FcRn(Kim, J. K., et al., Eur. J. Immunol. 29 (1999) 2819-2855). ResiduesM252Y, S254T, T256E have been described by Dall'Acqua et al. to improveFcRn binding by protein-protein interaction studies (Dall'Acqua, W. F.,et al. J. Biol. Chem. 281 (2006) 23514-23524). Studies of the humanFc-human FcRn complex have shown that residues I253, S254, H435, andY436 are crucial for the interaction (Firan, M., et al., Int. Immunol.13 (2001) 993-1002; Shields, R. L., et al., J. Biol. Chem. 276 (2001)6591-6604). In Yeung, Y. A., et al. (J. Immunol. 182 (2009) 7667-7671)various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and424 to 437 have been reported and examined. Exemplary mutations andtheir effect on FcRn binding are listed in the following Table.

effect on FcRn half-live in the mutation binding circulation referenceH285 reduced reduced Kim, J. K., H310Q/H433N (murine) (in mouse) Scand.J. (murine IgG1) Immunol. 40 (1994) 457-465 I253A reduced reducedGhetie, V. and H310A (murine) (in mouse) Ward, E. S., H435A Immunol.H436A Today 18 (murine IgG1) (1997) 592-598 T252L/T254S/T256F increasedincreased Ghetie, V. and T252A/T254S/T256A (murine) (in mouse) Ward, E.S., (murine IgG1) Immunol. Today 18 (1997) 592-598 I253A reduced reducedMedesan, C., H310A (murine) (in mouse) et al., J. H435A Immunol. 158H436A (1997) 2211-2217 H433A/N434Q (murine IgG1) I253A reduced reducedKim, J. K., H310A H310A: <0.1 (in mouse) Eur. J. H435A rel. binding toImmunol. 29 H435R muFcRn (1999) 2819-2825 (human IgG1) (murine) H433A1.1 rel. binding Kim, J. K., (human IgG1) to muFcRn, Eur. J. 0.4 rel.binding Immunol. 29 hu FcRn (1999) 2819-2825 (murine) I253A reducedreduced Shields, R. L., S254A <0.1 relative et al., J. Biol. H435Abinding to Chem. 276 Y436A huFcRn (2001) 6591-6604 (human IgG1) R255Areduced reduced Shields, R. L., K288A (human) et al., J. Biol. L309AChem. 276 S415A (2001) 6591-6604 H433A (human IgG1) P238A increasedincreased Shields, R. L., T256A (human) et al., J. Biol. E272A Chem. 276V305A (2001) 6591-6604 T307A Q311A D312A K317A D376A A378Q E380A E382AS424A N434A K288A/N434A E380A/N434A T307A/E380A/N434A (human IgG1) H435Areduced reduced Firan, M., et (humanized IgG1) <0.1 rel. al., Int.binding to Immunol. 13 huFcRn (2001) 993-1002 I253A (no binding)increased reduced Dall'Acqua, J. M252W (murine and (in mouse) Immunol.169 M252Y human) (2002) 5171-5180 M252Y/T256Q M252F/T256D N434F/Y436HM252Y/S254T/T256E G385A/Q386P/N389S H433K/N434F/Y436H H433R/N434Y/Y436HG385R/Q386T/P387R/N389P M252Y/S254T/T256E/H433K/ N434F/Y436HM252Y/S254T/T256E/G385R/ Q386T/P387R/N389P (human IgG1) M428L increasedincreased Hinton, P. R., T250Q/M428L (human) (in monkey) et al., J.Biol. (human IgG2) Chem. 279 (2004) 6213-6216 M252Y/S254T/T256E +increased increased Vaccaro, C., et H433K/N434F (human) (in mouse) al.,Nat. (human IgG) Biotechnol. 23 (2005) 1283-1288 T307A/E380A/N434Aincreased increased in Pop, L. M., et (chimeric IgG1) transgenic mouseal., Int. Immunopharmacol. 5 (2005) 1279-1290 T250Q increased increasedin Petkova, S. B., E380A (human) transgenic mouse et al., Int. M428LImmunol 18 N434A (2006) 1759-1769 K288A/N434A E380A/N434AT307A/E380A/N434A (human IgG1) I253A reduced reduced in Petkova, S. B.,(human IgG1) (human) transgenic mouse et al., Int. Immunol 18 (2006)1759-1769 S239D/A330L/I332E increased increased in Dall'Acqua, W. F.,M252Y/S254T/T256E (human and Cynomolgus et al., J. (humanized)Cynomolgus) Biol. Chem. 281 (2006) 23514-23524 T250Q increased increasedin Rhesus Hinton, P. R., M428L (human) apes et al., J. T250Q/M428LImmunol. 176 (human IgG1) (2006) 346-356 T250Q/M428L increased no changein Datta- P257I/Q311I (mouse and Cynomolgus Mannan, A., et (humanizedIgG1) Cynomolgus) increased in mouse al., J. Biol. Chem. 282 (2007)1709-1717 P257I/Q311I increased reduced in mice Datta- P257I/N434H at pH6 P257I/N434H Mannan, A., et D376V/N434H (human, reduced in al., Drug(humanized IgG1) Cynomolgus, Cynomolgus Metab. mouse) Dispos. 35 (2007)86-94 abrogate FcRn binding: increased and reducing the Ropeenian, D. C.I253 reduced binding ability of and H310 IgG for FcRn Akilesh, S., H433reduces its serum Nat. Rev. H435 persistence; a Immunol. 7 reduce FcRnbinding: higher-affinity (2007) 715-725 Y436 FcRn-IgG increased FcRnbinding: interaction prolongs T250 the half-lives of IgG N252 andFc-coupled S254 drugs in the serum T256 T307 M428 N434 N434A increasedincreased in Yeung, Y. A., T307Q/N434A (Cynomolgus Cynomolgus et al.,Cancer T307Q/N434S monkey) monkey Res. 70 (2010) V308P/N434A 3269-3277T307Q/E380A/N434A (human IgG1) 256P increased at WO 2011/ 280K neutralpH 122011 339T 385H 428L 434W/Y/F/A/H (human IgG)

It has been found that one mutation, one-sided in one Fc-regionpolypeptide, is sufficient to weaken the binding significantly. The moremutations that are introduced into the Fc-region, the weaker the bindingto the FcRn becomes. But one-sided asymmetric mutations are notsufficient to completely inhibit FcRn binding. Mutations on both sidesare necessary to completely inhibit FcRn binding.

The results of a symmetric engineering of an IgG1 Fc-region to influenceFcRn binding is shown in the following table (alignment of mutations andretention time on an FcRn-affinity chromatography column).

FcRn- affinity FcRn- FcRn- FcRn- column effector function bindingbinding binding retention influencing influencing influencinginfluencing time mutations mutation 1 mutation 2 mutation 3 [min]L234A/L235A/ — — — 45.3 P329G L234A/L235A/ I253A H310A H435A 2.3 P329GL234A/L235A/ I253A — — 2.7 P329G L234A/L235A/ — H310A — 2.4 P329GL234A/L235A/ — — H435A 2.7 P329G L234A/L235A/ I253A H310A — 2.3 P329GL234A/L235A/ I253A — H435A 2.3 P329G L234A/L235A/ — H310A H435A 2.4P329G L234A/L235A/ — H310A Y436A 2.3 P329G L234A/L235A/ H310A H433AY436A 2.4 P329G L234A/L235A/ — — Y436A 41.3 P329G

Retention times below 3 minutes correspond to no binding as thesubstance is in the flow-through (void peak).

The single mutation H310A is the most silent symmetrical mutation todelete any FcRn-binding.

The symmetric single mutation I253A and H435A result in a relative shiftof retention time of 0.3 to 0.4 min. This can be generally regarded as anon-detectable binding.

The single mutation Y436A results in detectable interaction strength tothe FcRn affinity column. Without being bound by theory this mutationcould have an effect on FcRn mediated half-life in vivo, which can bedifferentiated from a zero interaction such as the combination of theI253A, H310A and H435A mutations (IHH-AAA mutation).

The results obtained with a symmetrically modified anti-HER2 antibodyare presented in the following table (see WO 2006/031370 for reference).

retention time mutation [min] I253H no binding M252D no binding S254D nobinding R255D 41.4 M252H 43.6 K288E 45.2 L309H 45.5 E258H 45.6 T256H46.0 K290H 46.2 D98E 46.2 wild-type 46.3 K317H 46.3 Q311H 46.3 E430H46.4 T307H 47.0 N434H 52.0

The effect of the introduction of asymmetric FcRn-binding affectingmutations in the Fc-region has been exemplified with a bispecificantibody assembled using the knobs-into-holes technology (see e.g. U.S.Pat. No. 7,695,936, US 2003/0078385; “hole chain” mutations:S354C/T366W, “knob chain” mutations: Y349C/T366S/L368A/Y407V). Theeffect of the asymmetrically introduced mutations on FcRn-binding caneasily be determined using an FcRn affinity chromatography method (seeFIG. 9 and the following Table). Antibodies that have a later elutionfrom the FcRn affinity column, i.e. that have a longer retention time onthe FcRn affinity column, have a longer half-life in vivo, and viceversa.

retention time on FcRn affecting mutation FcRn affinity column one chainwith M252Y/S254T/T256E 56.2 min. none 51.8 min. one chain with I253A orH435A 48.8 min. one chain with H310A 48.4 min. one chain withI253A/H435A or I253A/H310A 48.0 min. or H310A/H435A one chain withH310A/H433A/Y436A 46.7 min. one chain with I253A/H310A/H435A 46.6 min.one chain with L251D/L314D/L432D 46.3 min. first chain withI253A/H310A/H435A and second no binding chain with H310A or H435A orI253A/H310A/H435A

The effect of the introduction of asymmetric FcRn-binding affectingmutations in the Fc-region has further been exemplified with amonospecific anti-IGF-1R antibody assembled using the knobs-into-holestechnology in order to allow the introduction of asymmetric mutations(see e.g. U.S. Pat. No. 7,695,936, US 2003/0078385; “hole chain”mutations: S354C/T366W, “knob chain” mutations:Y349C/T366S/L368A/Y407V). The effect of the asymmetrically introducedmutations on FcRn-binding can easily be determined using an FcRnaffinity chromatography method (see the following Table). Antibodiesthat have a later elution from the FcRn affinity column, i.e. that havea longer retention time on the FcRn affinity column, have a longerhalf-life in vivo, and vice versa.

retention time on FcRn affecting mutation FcRn affinity column one chainwith M252Y/S254T/T256E 57.6 min. none 53.0 min. one chain withH310A/H433A/Y436A 42.4 min. one chain with I253A/H310A/H435A 42.0 min.one chain with L251D/L314D/L432D 40.9 min. first chain withI253A/H310A/H435A and second no binding chain with H310A or H435A orI253A/H310A/H435A

The asymmetric IHH-AAA and LLL-DDD mutations (LLL-DDDmutation=combination of the mutations L251D, L314D and L432D) showweaker binding than the corresponding parent or wild-type antibody.

The symmetric HHY-AAA mutation (=combination of the mutations H310A,H433A and Y436A) results in an Fc-region that does no longer bind to thehuman FcRn whereas the binding to protein A is maintained (see FIGS. 11,12, 13 and 14 ).

The effect of the introduction of asymmetric FcRn-binding affectingmutations in the Fc-region has further been exemplified with amonospecific anti-IGF-1R antibody (IGF-1R), a bispecific anti-VEGF/ANG2antibody (VEGF/ANG2), and a full length antibody with fusions to theC-terminus of both heavy chains (fusion) assembled using theknobs-into-holes technology in order to allow the introduction ofasymmetric mutations (see e.g. U.S. Pat. No. 7,695,936, US 2003/0078385;“hole chain” mutations: S354C/T366W, “knob chain” mutations:Y349C/T366S/L368A/Y407V). The effect of the introduced mutations onFcRn-binding and protein A binding can easily be determined using anFcRn affinity chromatography method, a protein A affinity chromatographymethod and SPR-based methods (see the following Table).

protein further further FcR A muta- muta- binding FcRn protein bind-tion tion affecting FcRn binding A ing anti- in knob in hole muta-binding (col- binding (col- body chain chain tions (SPR) umn) (SPR) umn)VEGF/ none none L234A yes yes stable yes ANG2 L235A binding 0096 P329GVEGF/ none I253A L234A yes yes fast yes ANG2 H310A L235A off- 0097 H435AP329G rate VEGF/ none H310A L234A yes yes stable yes ANG2 H433A L235Abinding 0098 Y436A P329G VEGF/ none L251D L234A re- re- fast yes ANG2L314D L235A duced duced off- 0099 L432D P329G rate VEGF/ none M252YL234A in- in- n.d. yes ANG2 S254T L235A creased creased 0100 T256E P329GVEGF/ I253A I253A L234A n.d. no n.d. no ANG2 H310A H310A L235A 0016H435A H435A P329G VEGF/ H310A H310A L234A n.d. n.d. n.d. yes ANG2 H433AH433A L235A 0121 Y436A Y436A P329G IGF-1R none none n.d. yes 0033 IGF-none I253A L234A n.d. yes n.d. yes 1R H310A L235A 0034 H435A P329G IGF-none H310A none re- re- n.d. yes 1R H433A duced duced 0035 Y436A IGF-none L251D L234A n.d. yes n.d. yes 1R L314D L235A 0037 L432D P329G IGF-none M252Y L234A n.d. yes n.d. yes 1R S254T L235A 0036 T256E P329G IGF-H310A H310A none n.d. n.d. n.d. yes 1R H433A H433A 0045 Y436A Y436Afusion none none L234A n.d. yes n.d. n.d. 0008 L235A P329G fusion I253AI253A L234A n.d. no n.d. n.d. 0019 L235A P329G fusion H310A H310A L234An.d. no n.d. n.d. 0020 L235A P329G fusion H435A H435A L234A n.d. no n.d.n.d. 0021 L235A P329G fusion Y436A Y436A L234A n.d. re- n.d. n.d. 0038L235A duced P329G fusion I253A I253A L234A n.d. no n.d. n.d. 0022 H310AH310A L235A P329G fusion I253A I253A L234A n.d. no n.d. n.d. 0023 H435AH435A L235A P329G fusion H310A H310A L234A n.d. no n.d. n.d. 0036 H435AH435A L235A P329G fusion H310A H310A L234A n.d. no n.d. n.d. 0037 Y436AY436A L235A P329G fusion 1253A I253A L234A n.d. no n.d. n.d. 0018 H310AH310A L235A H435A H435A P329G fusion H310A H310A L234A n.d. no n.d. n.d.0019 H433A H433A L235A Y436A Y436A P329G

One aspect as reported herein is an antibody or Fc-region fusionpolypeptide comprising the variant human IgG class Fc-region as reportedherein.

The Fc-region (dimeric polypeptide) as reported herein, when containedin an Fc-region fusion polypeptide or a full length antibody confers theabove described characteristics to the molecule. The fusion partner canbe any molecule having a biological activity who's in vivo half-liveshall be reduced or increased, i.e. who's in vivo half-live shall beclearly defined and tailor-made for its intended application.

Fc-region fusion polypeptides may comprise e.g. a variant (human) IgGclass Fc-region as reported herein and a receptor protein that binds toa target including a ligand, such as, for example, TNFR-Fc-region fusionpolypeptide (TNFR=human tumor necrosis factor receptor), orIL-1R-Fc-region fusion polypeptide (IL-1R=human interleukin-1 receptor),or VEGFR-Fc-region fusion polypeptides (VEGFR=human vascular endothelialgrowth factor receptor), or ANG2R-Fc-region fusion polypeptides(ANG2R=human angiopoietin 2 receptor).

Fc-region fusion polypeptides may comprise e.g. a variant (human) IgGclass Fc-region as reported herein and an antibody fragment that bindsto a target including, such as, for example, an antibody Fab fragment,scFvs (see e.g. Nat. Biotechnol. 23 (2005) 1126-1136), or domainantibodies (dAbs) (see e.g. WO 2004/058821, WO 2003/002609).

Fc-region fusion polypeptides may comprise e.g. a variant (human) humanIgG class Fc-region as reported herein and a receptor ligand (eithernaturally occurring or artificial).

Antibodies, e.g. full length antibodies or CrossMabs, can comprise avariant (human) human IgG class Fc-region as reported herein.

B. Ocular Vascular Diseases

Ocular vascular diseases are any pathological condition characterized byaltered or unregulated proliferation and invasion of new blood vesselsinto the structures of ocular tissues such as the retina or cornea.

In one embodiment the ocular vascular disease is selected from the groupconsisting of wet age-related macular degeneration (wet AMD), dryage-related macular degeneration (dry AMD), diabetic macular edema(DME), cystoid macular edema (CME), non-proliferative diabeticretinopathy (NPDR), proliferative diabetic retinopathy (PDR), cystoidmacular edema, vasculitis (e.g. central retinal vein occlusion),papilloedema, retinitis, conjunctivitis, uveitis, choroiditis,multifocal choroiditis, ocular histoplasmosis, blepharitis, dry eye(Sjogren's disease), and other ophthalmic diseases wherein the eyedisease or disorder is associated with ocular neovascularization,vascular leakage, and/or retinal edema.

The antibody comprising the dimeric polypeptide as reported herein isuseful in the prevention and treatment of wet AMD, dry AMD, CME, DME,NPDR, PDR, blepharitis, dry eye and uveitis, in one preferred embodimentwet AMD, dry AMD, blepharitis, and dry eye, also in one preferredembodiment CME, DME, NPDR and PDR, also in one preferred embodimentblepharitis, and dry eye, in particular wet AMD and dry AMD, and alsoparticularly wet AMD.

In some embodiments, the ocular vascular disease is selected from thegroup consisting of wet age-related macular degeneration (wet AMD),macular edema, retinal vein occlusions, retinopathy of prematurity, anddiabetic retinopathy.

Other diseases associated with corneal neovascularization include, butare not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency,contact lens overwear, atopic keratitis, superior limbic keratitis,pterygium keratitis sicca, Sjogren's disease, acne rosacea,phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration,chemical burns, bacterial ulcers, fungal ulcers, Herpes simplexinfections, Herpes zoster infections, protozoan infections, Kaposisarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginalkeratolysis, rheumatoid arthritis, systemic lupus, polyarteritis,trauma, Wegener's sarcoidosis, Scleritis, Steven's Johnson disease,periphigoid radial keratotomy, and corneal graph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget'sdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, retinitispigmentosa, retina edema (including macular edema), Eale's disease,Bechet's disease, infections causing a retinitis or choroiditis,presumed ocular histoplasmosis, Best's disease, myopia, optic pits,Stargart's disease, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, trauma, and post-lasercomplications.

Other diseases include, but are not limited to, diseases associated withrubeosis (neovascularization of the angle) and diseases caused by theabnormal proliferation of fibrovascular or fibrous tissue including allforms of proliferative vitreoretinopathy.

Retinopathy of prematurity (ROP) is a disease of the eye that affectsprematurely born babies. It is thought to be caused by disorganizedgrowth of retinal blood vessels which may result in scarring and retinaldetachment. ROP can be mild and may resolve spontaneously, but may leadto blindness in serious cases. As such, all preterm babies are at riskfor ROP, and very low birth weight is an additional risk factor. Bothoxygen toxicity and relative hypoxia can contribute to the developmentof ROP.

Macular degeneration is a medical condition predominantly found inelderly adults in which the center of the inner lining of the eye, knownas the macula area of the retina, suffers thinning, atrophy, and in somecases, bleeding. This can result in loss of central vision, whichentails inability to see fine details, to read, or to recognize faces.According to the American Academy of Ophthalmology, it is the leadingcause of central vision loss (blindness) in the United States today forthose over the age of fifty years. Although some macular dystrophiesthat affect younger individuals are sometimes referred to as maculardegeneration, the term generally refers to age-related maculardegeneration (AMD or ARMD).

Age-related macular degeneration begins with characteristic yellowdeposits in the macula (central area of the retina which providesdetailed central vision, called fovea) called drusen between the retinalpigment epithelium and the underlying choroid. Most people with theseearly changes (referred to as age-related maculopathy) have good vision.People with drusen can go on to develop advanced AMD. The risk isconsiderably higher when the drusen are large and numerous andassociated with disturbance in the pigmented cell layer under themacula. Large and soft drusen are related to elevated cholesteroldeposits and may respond to cholesterol lowering agents or the RheoProcedure.

Advanced AMD, which is responsible for profound vision loss, has twoforms: dry and wet. Central geographic atrophy, the dry form of advancedAMD, results from atrophy to the retinal pigment epithelial layer belowthe retina, which causes vision loss through loss of photoreceptors(rods and cones) in the central part of the eye. While no treatment isavailable for this condition, vitamin supplements with high doses ofantioxidants, lutein and zeaxanthin, have been demonstrated by theNational Eye Institute and others to slow the progression of dry maculardegeneration and in some patients, improve visual acuity.

Retinitis pigmentosa (RP) is a group of genetic eye conditions. In theprogression of symptoms for RP, night blindness generally precedestunnel vision by years or even decades. Many people with RP do notbecome legally blind until their 40s or 50s and retain some sight alltheir life. Others go completely blind from RP, in some cases as earlyas childhood. Progression of RP is different in each case. RP is a typeof hereditary retinal dystrophy, a group of inherited disorders in whichabnormalities of the photoreceptors (rods and cones) or the retinalpigment epithelium (RPE) of the retina lead to progressive visual loss.Affected individuals first experience defective dark adaptation ornyctalopia (night blindness), followed by reduction of the peripheralvisual field (known as tunnel vision) and, sometimes, loss of centralvision late in the course of the disease.

Macular edema occurs when fluid and protein deposits collect on or underthe macula of the eye, a yellow central area of the retina, causing itto thicken and swell. The swelling may distort a person's centralvision, as the macula is near the center of the retina at the back ofthe eyeball. This area holds tightly packed cones that provide sharp,clear central vision to enable a person to see form, color, and detailthat is directly in the line of sight. Cystoid macular edema is a typeof macular edema that includes cyst formation.

C. Antibody Purification with a Staphylococcus Protein a AffinityChromatography Column

In one aspect, a dimeric polypeptide comprising

-   -   a first polypeptide and a second polypeptide each comprising in        N-terminal to C-terminal direction at least a portion of an        immunoglobulin hinge region, which comprises one or more        cysteine residues, an immunoglobulin CH2-domain, and an        immunoglobulin CH3-domain,    -   wherein the first, the second, or the first and the second        polypeptide comprise the mutation Y436A (numbering according to        the Kabat EU index numbering system)        is provided.

This dimeric polypeptide has, due to the mutation, the properties ofimproved binding to Staphylococcal protein A.

Thus, these antibodies can be purified, i.e. separated from unwantedby-products by using conventional protein A affinity materials, such asMABSELECTSURE™. It is not required to use highly sophisticated butspecies limited affinity materials, such as e.g. KappaSelect, which isonly useable with antibodies comprising a light chain of the kappasubclass. Additionally it is not required to adopt the purificationmethod if a modification/exchange of the light chain subclass is made(see FIGS. 11 and 12 , respectively).

One aspect as reported herein is a method for producing a dimericpolypeptide as reported herein comprising the following steps:

-   -   a) cultivating a mammalian cell comprising one or more nucleic        acids encoding a dimeric polypeptide as reported herein,    -   b) recovering the dimeric polypeptide from the cultivation        medium, and    -   c) purifying the dimeric polypeptide with a protein A affinity        chromatography and thereby producing the dimeric polypeptide.

One aspect as reported herein is the use of the mutation Y436A forincreasing the binding of a dimeric Fc-region polypeptide to protein A.

It has been found that by introducing the mutation Y436A, the binding toStaphylococcal protein A (SPA) can be increased. This is advantageouse.g. if additional mutations are introduced that reduce the binding toSPA, such as e.g. I253A and H310A or H310A and H435A (see FIG. 15 ).

The dimeric polypeptide as reported herein is produced by recombinantmeans. Thus, one aspect of the current invention is a nucleic acidencoding the dimeric polypeptide as reported herein and a further aspectis a cell comprising the nucleic acid encoding the dimeric polypeptideas reported herein. Methods for recombinant production are widely knownin the state of the art and comprise protein expression in prokaryoticand eukaryotic cells with subsequent isolation of the dimericpolypeptide and usually purification to a pharmaceutically acceptablepurity. For the expression of the dimeric polypeptides as aforementionedin a host cell, nucleic acids encoding the respective first and secondpolypeptides are inserted into expression vectors by standard methods.Expression is performed in appropriate prokaryotic or eukaryotic hostcells like CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells,PER.C6 cells, yeast, or E. coli cells, and the dimeric polypeptide isrecovered from the cells (cultivation supernatant or cells after lysis).

General methods for recombinant production of antibodies are well-knownin the state of the art and described, for example, in the reviewarticles of Makrides, S. C., Protein Expr. Purif. 17 (1999) 183-202;Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-160; Werner, R. G., Drug Res. 48(1998) 870-880.

Accordingly, one aspect as reported herein is a method for theproduction of a dimeric polypeptide as reported herein, comprising thesteps of

-   -   a) transforming a host cell with one or more vectors comprising        nucleic acid molecules encoding a dimeric polypeptide as        reported herein,    -   b) culturing the host cell under conditions that allow synthesis        of the dimeric polypeptide, and    -   c) recovering the dimeric polypeptide from the culture and        thereby producing the dimeric polypeptide.

In one embodiment the recovering step under c) includes the use of animmunoglobulin Fc-region specific capture reagent. In one embodimentthis Fc-region specific capture reagent is used in abind-and-elute-mode). Examples of such Fc-region specific capturereagents are e.g. Staphylococcus protein A-based affinity chromatographycolumns, which are based on a highly rigid agarose base matrix thatallows high flow rates and low back pressure at large scale. Theyfeature a ligand that binds to the dimeric polypeptide, i.e. itsFc-region. The ligands are attached to the matrix via a long hydrophilicspacer arm to make it easily available for binding to the targetmolecule.

The dimeric polypeptides as reported herein are suitably separated fromthe culture medium by conventional immunoglobulin purificationprocedures such as, for example, protein A-Sepharose, hydroxylapatitechromatography, gel electrophoresis, dialysis, or affinitychromatography. B-cells or hybridoma cells can serve as a source of DNAand RNA encoding the dimeric polypeptide. DNA and RNA encoding themonoclonal antibodies are readily isolated and sequenced usingconventional procedures. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise producedimeric polypeptides, to obtain the synthesis of recombinant monoclonaldimeric polypeptides in the host cells.

Purification of antibodies is performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art (see Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987)). Different methods are well established and in widespread usefor protein purification, such as affinity chromatography with microbialproteins (e.g. protein A or protein G affinity chromatography), ionexchange chromatography (e.g. cation exchange (carboxymethyl resins),anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75(1998) 93-102).

One aspect of the invention is a pharmaceutical formulation comprising adimeric polypeptide or an antibody as reported herein. Another aspect ofthe invention is the use of a dimeric polypeptide or an antibody asreported herein for the manufacture of a pharmaceutical formulation. Afurther aspect of the invention is a method for the manufacture of apharmaceutical formulation comprising a dimeric polypeptide or anantibody as reported herein. In another aspect, the present inventionprovides a formulation, e.g. a pharmaceutical formulation, containing adimeric polypeptide or an antibody as reported herein, formulatedtogether with a pharmaceutical carrier.

A formulation as reported herein can be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. To administer a compound of the invention by certainroutes of administration, it may be necessary to coat the compound with,or co-administer the compound with, a material to prevent itsinactivation. For example, the compound may be administered to a subjectin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

Many possible modes of delivery can be used, including, but not limitedto intraocular application or topical application. In one embodiment theapplication is intraocular and includes, but is not limited to,subconjunctival injection, intracanieral injection, injection into theanterior chamber via the termporai limbus, intrastromal injection,intracorneal injection, subretinal injection, aqueous humor injection,subtenon injection or sustained delivery device, intravitreal injection(e.g., front, mid or back vitreal injection). In one embodiment theapplication is topical and includes, but is not limited to, eye drops tothe cornea.

In one embodiment the dimeric polypeptide as reported herein or thepharmaceutical formulation as reported herein is administered viaintravitreal application, e.g. via intravitreal injection. This can beperformed in accordance with standard procedures known in the art (see,e.g., Ritter et al., J. Clin. Invest. 116 (2006) 3266-3276;Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999)196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-185).

In some embodiments, therapeutic kits of the invention can contain oneor more doses of a dimeric polypeptide as reported herein present in apharmaceutical formulation as described herein, a suitable device forintravitreal injection of the pharmaceutical formulation, and aninstruction detailing suitable subjects and protocols for carrying outthe injection. In these embodiments, the formulations are typicallyadministered to the subject in need of treatment via intravitrealinjection. This can be performed in accordance with standard proceduresknown in the art. See, e.g., Ritter et al., J. Clin. Invest. 116 (2006)3266-3276; Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25(1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-185.

The formulation may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe presence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the formulations. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds asreported herein, which may be used in a suitable hydrated form, and/orthe pharmaceutical formulations as reported herein, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalformulation as reported herein may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health, and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The formulation must be sterile and fluid to the extent that theformulation is deliverable by syringe. In addition to water, the carrierin one preferred embodiment is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

The formulation can comprise an ophthalmic depot formulation comprisingan active agent for subconjunctival administration. The ophthalmic depotformulation comprises microparticles of essentially pure active agent,e.g., a dimeric polypeptide as reported herein. The microparticlescomprising a dimeric polypeptide as reported herein can be embedded in abiocompatible pharmaceutically acceptable polymer or a lipidencapsulating agent. The depot formulations may be adapted to releaseall, or substantially all, the active material over an extended periodof time. The polymer or lipid matrix, if present, may be adapted todegrade sufficiently to be transported from the site of administrationafter release of all or substantially all the active agent. The depotformulation can be liquid formulation, comprising a pharmaceuticalacceptable polymer and a dissolved or dispersed active agent. Uponinjection, the polymer forms a depot at the injections site, e.g. bygelifying or precipitating.

Another aspect of the invention is a dimeric polypeptide or an antibodyas reported herein for use in the treatment of ocular vascular diseases.

One embodiment of the invention is a dimeric polypeptide or an antibodyas reported herein for use in the treatment of ocular vascular diseases.

Another aspect of the invention is the pharmaceutical formulation foruse in the treatment of ocular vascular diseases.

Another aspect of the invention is the use of a dimeric polypeptide oran antibody as reported herein for the manufacture of a medicament forthe treatment of ocular vascular disease.

Another aspect of the invention is method of treating a patientsuffering from ocular vascular diseases by administering a dimericpolypeptide or an antibody as reported herein to a patient in the needof such treatment.

It is herewith expressly stated that the term “comprising” as usedherein comprises the term “consisting of.” Thus, all aspects andembodiments that contain the term “comprising” are likewise disclosedwith the term “consisting of.”

D. Modifications

In a further aspect, a dimeric polypeptide according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-6 below:

1. Antibody Affinity

In one embodiment, Kd is measured using a BIACORE® surface plasmonresonance assay. For example, an assay using a BIACORE®-2000 or aBIACORE®-3000 (GE Healthcare Inc., Piscataway, N.J.) is performed at 25°C. with immobilized binding partner CM5 chips at ˜10 response units(RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5,GE Healthcare Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NETS) according to the supplier's instructions.The binding partner is diluted with 10 mM sodium acetate, pH 4.8, to 5μg/mL (˜0.2 μM) before injection at a flow rate of 5 μl/minute toachieve approximately 10 response units (RU) of coupled binding partner.Following the injection of the binding partner, 1 M ethanolamine isinjected to block non-reacted groups. For kinetics measurements,two-fold serial dilutions of the dimeric polypeptide containing fusionpolypeptide or antibody (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μL/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on) (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999)865-881). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Chimeric and Humanized Antibodies

In certain embodiments, a dimeric polypeptide as reported herein is achimeric antibody. Certain chimeric antibodies are described, e.g., inU.S. Pat. No. 4,816,567; and Morrison, S. L., et al., Proc. Natl. Acad.Sci. USA 81 (1984) 6851-6855). In one example, a chimeric antibodycomprises a non-human variable region (e.g., a variable region derivedfrom a mouse, rat, hamster, rabbit, or non-human primate, such as amonkey) and a human constant region. In a further example, a chimericantibody is a “class switched” antibody in which the class or subclasshas been changed from that of the parent antibody. Chimeric antibodiesinclude antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I., et al., Nature 332 (1988)323-329; Queen, C., et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5,821,337; 7,527,791; 6,982,321; and7,087,409; Kashmiri, S. V., et al., Methods 36 (2005) 25-34 (describingspecificity determining region (SDR) grafting); Padlan, E. A., Mol.Immunol. 28 (1991) 489-498 (describing “resurfacing”); Dall'Acqua, W. F.et al., Methods 36 (2005) 43-60 (describing “FR shuffling”); Osbourn, J.et al., Methods 36 (2005) 61-68; and Klimka, A. et al., Br. J. Cancer 83(2000) 252-260 (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, M. J., et al., J. Immunol. 151 (1993)2296-2308; framework regions derived from the consensus sequence ofhuman antibodies of a particular subgroup of light or heavy chainvariable regions (see, e.g., Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Presta, L. G., et al., J. Immunol. 151(1993) 2623-2632); human mature (somatically mutated) framework regionsor human germline framework regions (see, e.g., Almagro, J. C. andFransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regionsderived from screening FR libraries (see, e.g., Baca, M. et al., J.Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol.Chem. 271 (19969 22611-22618).

3. Human Antibodies

In certain embodiments, a dimeric polypeptide as reported herein is ahuman antibody. Human antibodies can be produced using varioustechniques known in the art. Human antibodies are described generally invan Dijk, M. A. and van de Winkel, J. G., Curr. Opin. Pharmacol. 5(2001) 368-374, and Lonberg, N., Curr. Opin. Immunol. 20 (2008) 450-459.

Human antibodies maybe prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125.See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE′ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and US 2007/0061900, describing VELOCIMOUSE® technology). Human variableregions from intact antibodies generated by such animals may be furthermodified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor, D.,J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R., et al., MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York (1987), pp. 51-63; and Boerner, P., et al., J. Immunol. 147(1991) 86-95). Human antibodies generated via human B-cell hybridomatechnology are also described in Li, J., et al., Proc. Natl. Acad. Sci.USA 103 (2006) 3557-3562. Additional methods include those described,for example, in U.S. Pat. No. 7,189,826 (describing production ofmonoclonal human IgM antibodies from hybridoma cell lines); Ni, J.,Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas).Human hybridoma technology (Trioma technology) is also described inVollmers, H. P. and Brandlein, S., Histology and Histopathology 20(2005) 927-937; and Vollmers, H. P. and Brandlein, S., Methods andFindings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

4. Library-Derived Antibodies

In certain embodiments a dimeric polypeptide as reported herein is alibrary-derived antibody. Library-derived antibodies may be isolated byscreening combinatorial libraries for antibodies with the desiredactivity or activities. For example, a variety of methods are known inthe art for generating phage display libraries and screening suchlibraries for antibodies possessing the desired binding characteristics.Such methods are reviewed, e.g., in Hoogenboom, H. R. et al., Methods inMolecular Biology 178 (2001) 1-37 and further described, e.g., in theMcCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T. et al.,Nature 352 (1991) 624-628; Marks, J. D. et al., J. Mol. Biol. 222 (1992)581-597; Marks, J. D. and Bradbury, A., Methods in Molecular Biology 248(2003) 161-175; Sidhu, S. S. et al., J. Mol. Biol. 338 (2004) 299-310;Lee, C. V. et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, F. A.,Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472; and Lee, C. V. etal., J. Immunol. Methods 284 (2004) 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter, G., et al., Ann. Rev.Immunol. 12 (1994) 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen without the requirement of constructing hybridomas.Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self-antigens without any immunization as described by Griffiths,A. D., et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries canalso be made synthetically by cloning non-rearranged V-gene segmentsfrom stem cells, and using PCR primers containing random sequence toencode the highly variable CDR3 regions and to accomplish rearrangementin vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.Biol. 227 (1992) 381-388. Patent publications describing human antibodyphage libraries include, for example: U.S. Pat. No. 5,750,373, and US2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US2007/0160598, US 2007/0237764, US 2007/0292936, and US 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

5. Multispecific Antibodies

In certain embodiments, a dimeric polypeptide as reported herein is amultispecific antibody, e.g. a bispecific antibody. Multispecificantibodies are monoclonal antibodies that have binding specificities forat least two different sites. In certain embodiments, one of the bindingspecificities is for a first antigen and the other is for a different,second antigen. In certain embodiments, bispecific antibodies may bindto two different epitopes of the same antigen. Bispecific antibodies mayalso be used to localize cytotoxic agents to cells which express atleast one of the antigens. Bispecific antibodies can be prepared as fulllength antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein, C.and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, andTraunecker, A., et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole”engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelny,S. A., et al., J. Immunol. 148 (1992) 1547-1553; using “diabody”technology for making bispecific antibody fragments (see, e.g.,Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448);and using single-chain Fv (scFv) dimers (see, e.g. Gruber, M et al., J.Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies asdescribed, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576).

The antibody or fragment herein also includes a “Dual Acting Fab” or“DAF” (see, US 2008/0069820, for example).

The antibody or fragment herein also includes multispecific antibodiesdescribed in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO2010/145792, and WO 2010/145793.

6. Antibody Variants

In certain embodiments, a dimeric polypeptide as reported herein is anantibody. In further embodiments amino acid sequence variants of theantibodies provided herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of an antibodymay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics, e.g.,antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in the Table below under the heading of “preferred substitutions.”More substantial changes are provided in the following Table under theheading of “exemplary substitutions,” and as further described below inreference to amino acid side chain classes. Amino acid substitutions maybe introduced into an antibody of interest and the products screened fora desired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

Original Exemplary Preferred Residue Substitutions Substitutions Ala (A)Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys;Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu AsnGlu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine; Ile;Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; IleLeu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) ThrThr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; SerPhe Val (V) Ile; Leu; Met; Phe; Ala; Leu Norleucine

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, P. S.,Methods Mol. Biol. 207 (2008) 179-196), and/or residues that contactantigen, with the resulting variant VH or VL being tested for bindingaffinity. Affinity maturation by constructing and reselecting fromsecondary libraries has been described, e.g., in Hoogenboom, H. R. etal. in Methods in Molecular Biology 178 (2002) 1-37. In some embodimentsof affinity maturation, diversity is introduced into the variable geneschosen for maturation by any of a variety of methods (e.g., error-pronePCR, chain shuffling, or oligonucleotide-directed mutagenesis). Asecondary library is then created. The library is then screened toidentify any antibody variants with the desired affinity. Another methodto introduce diversity involves HVR-directed approaches, in whichseveral HVR residues (e.g., 4-6 residues at a time) are randomized. HVRresidues involved in antigen binding may be specifically identified,e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two, or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham, B. C. and Wells, J. A., Science244 (1989) 1081-1085. In this method, a residue or group of targetresidues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu)are identified and replaced by a neutral or negatively charged aminoacid (e.g., alanine or polyalanine) to determine whether the interactionof the antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen can be used.Such contact residues and neighboring residues may be targeted oreliminated as candidates for substitution. Variants may be screened todetermine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc-region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of theFc-region. See, e.g., Wright, A. and Morrison, S. L., TIBTECH 15 (1997)26-32. The oligosaccharide may include various carbohydrates, e.g.,mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, aswell as a fucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to anFc-region. For example, the amount of fucose in such antibody may befrom 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. Theamount of fucose is determined by calculating the average amount offucose within the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc-region (EUnumbering of Fc-region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US 2003/0157108; US 2004/0093621. Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A.et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al.,Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable ofproducing defucosylated antibodies include Lec13 CHO cells deficient inprotein fucosylation (Ripka, J., et al., Arch. Biochem. Biophys. 249(1986) 533-545; US 2003/0157108; and WO 2004/056312, especially atExample 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki, N., et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda,Y., et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).

Antibody variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc-regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc-region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

c) Fc-Region Variants

In certain embodiments, one or more further amino acid modifications maybe introduced into a dimeric polypeptide as reported herein, therebygenerating an Fc-region variant. The Fc-region variant may comprise ahuman Fc-region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4Fc-region) comprising an amino acid modification (e.g. asubstitution/mutation) at one or more amino acid positions.

In certain embodiments, the invention contemplates a dimeric polypeptidethat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of thedimeric polypeptide in vivo is important yet certain effector functions(such as CDC and ADCC) are unnecessary or deleterious. In vitro and/orin vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that thedimeric polypeptide antibody lacks FcγR binding (hence likely lackingADCC activity), but retains FcRn binding ability. The primary cells formediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch, J. V. and Kinet, J. P.,Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitroassays to assess ADCC activity of a molecule of interest are describedin U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al., Proc. Natl.Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc.Natl. Acad. Sci. USA 82 (1985) 1499-1502); U.S. Pat. No. 5,821,337 (seeBruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361).Alternatively, non-radioactive assay methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes, R.et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assaysmay also be carried out to confirm that the dimeric polypeptide isunable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3cbinding ELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171;Cragg, M. S. et al., Blood 101 (2003) 1045-1052; and Cragg, M. S. and M.J. Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivoclearance/half-life determinations can also be performed using methodsknown in the art (see, e.g., Petkova, S. B. et al., Int. Immunol. 18(2006) 1759-1769).

Dimeric polypeptides with reduced effector function include those withsubstitution of one or more of Fc-region residues 238, 265, 269, 270,297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc-region variantsinclude Fc-regions with substitutions at two or more of amino acidpositions 265, 269, 270, 297, and 327, including the so-called “DANA”Fc-region mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604)

In certain embodiments, a dimeric polypeptide variant comprises anFc-region with one or more amino acid substitutions which improve ADCC,e.g., substitutions at positions 298, 333, and/or 334 of the Fc-region(EU numbering of residues).

In some embodiments, alterations are made in the Fc-region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164(2000) 4178-4184.

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593; and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc-regionwith one or more substitutions therein which improve binding of theFc-region to FcRn. Such Fc-region variants include those withsubstitutions at one or more of Fc-region residues: 238, 256, 265, 272,286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 413, 424 or 434, e.g., substitution of Fc-region residue 434 (U.S.Pat. No. 7,371,826).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc-region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered dimeric polypeptides, e.g., in analogy to “thioMAbs,” inwhich one or more residues of an antibody are substituted with cysteineresidues. In particular embodiments, the substituted residues occur ataccessible sites of the dimeric polypeptide. By substituting thoseresidues with cysteine, reactive thiol groups are thereby positioned ataccessible sites of the dimeric polypeptide and may be used to conjugatethe dimeric polypeptide to other moieties, such as drug moieties orlinker-drug moieties, to create an immunoconjugate, as described furtherherein. In certain embodiments, any one or more of the followingresidues may be substituted with cysteine: V205 (Kabat numbering) of thelight chain; A118 (EU numbering) of the heavy chain; and S400 (EUnumbering) of the heavy chain Fc-region. Cysteine engineered dimericpolypeptides may be generated as described, e.g., in U.S. Pat. No.7,521,541.

e) Derivatives

In certain embodiments, a dimeric polypeptide as reported herein may befurther modified to contain additional non-proteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the dimeric polypeptide include but are not limited towater soluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or non-branched. The number of polymersattached to the dimeric polypeptide may vary, and if more than onepolymer is attached, they can be the same or different molecules. Ingeneral, the number and/or type of polymers used for derivatization canbe determined based on considerations including, but not limited to, theparticular properties or functions of the dimeric polypeptide to beimproved, whether the dimeric polypeptide derivative will be used in atherapy under defined conditions, etc.

In another embodiment, conjugates of a dimeric polypeptide as reportedherein and non-proteinaceous moiety that may be selectively heated byexposure to radiation are provided. In one embodiment, thenon-proteinaceous moiety is a carbon nanotube (Kam, N. W. et al., Proc.Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation may be ofany wavelength, and includes, but is not limited to, wavelengths that donot harm ordinary cells, but which heat the non-proteinaceous moiety toa temperature at which cells proximal to the dimericpolypeptide-non-proteinaceous moiety are killed.

f) Heterodimerization

There exist several approaches for CH3-modifications to enforce theheterodimerization, which are well described e.g. in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO2013157954, WO 2013096291. Typically, in all such approaches the firstCH3 domain and the second CH3 domains are both engineered in acomplementary manner so that each CH3 domain (or the heavy chaincomprising it) can no longer homodimerize with itself but is forced toheterodimerize with the complementary-engineered other CH3 domain (sothat the first and second CH3 domain heterodimerize and no homodimersbetween the two first or the two second CH3 domains are formed). Thesedifferent approaches for improved heavy chain heterodimerization arecontemplated as different alternatives in combination with theheavy-light chain modifications (VH and VL exchange/replacement in onebinding arm and the introduction of substitutions of charged amino acidswith opposite charges in the CH1/CL interface) in the multispecificantibodies according to the invention which reduce light chainmispairing an Bence-Jones type side products.

In one preferred embodiment of the invention (in case the multispecificantibody comprises CH3 domains in the heavy chains) the CH3 domains ofsaid multispecific antibody according to the invention can be altered bythe “knob-into-holes” technology which is described in detail withseveral examples in e.g. WO 96/027011, Ridgway, J. B., et al., ProteinEng. 9 (1996) 617-621; and Merchant, A. M., et al., Nat. Biotechnol. 16(1998) 677-681; WO 98/050431. In this method the interaction surfaces ofthe two CH3 domains are altered to increase the heterodimerization ofboth heavy chains containing these two CH3 domains. Each of the two CH3domains (of the two heavy chains) can be the “knob”, while the other isthe “hole.” The introduction of a disulfide bridge further stabilizesthe heterodimers (Merchant, A. M., et al., Nature Biotech. 16 (1998)677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) andincreases the yield.

Thus in one embodiment of the invention said multispecific antibody(comprises a CH3 domain in each heavy chain and) is furthercharacterized in that

-   -   the first CH3 domain of the first heavy chain of the antibody        under a) and the second CH3 domain of the second heavy chain of        the antibody under b) each meet at an interface which comprises        an original interface between the antibody CH3 domains.        -   wherein said interface is altered to promote the formation            of the multispecific antibody, wherein the alteration is            characterized in that:        -   i) the CH3 domain of one heavy chain is altered,        -   so that within the original interface of the CH3 domain of            one heavy chain that meets the original interface of the CH3            domain of the other heavy chain within the multispecific            antibody,        -   an amino acid residue is replaced with an amino acid residue            having a larger side chain volume, thereby generating a            protuberance within the interface of the CH3 domain of one            heavy chain which is positionable in a cavity within the            interface of the CH3 domain of the other heavy chain        -   and        -   ii) the CH3 domain of the other heavy chain is altered,        -   so that within the original interface of the second CH3            domain that meets the original interface of the first CH3            domain within the multispecific antibody an amino acid            residue is replaced with an amino acid residue having a            smaller side chain volume, thereby generating a cavity            within the interface of the second CH3 domain within which a            protuberance within the interface of the first CH3 domain is            positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), valine (V).

In one aspect of the invention both CH3 domains are further altered bythe introduction of cysteine (C) as amino acid in the correspondingpositions of each CH3 domain such that a disulfide bridge between bothCH3 domains can be formed.

In one preferred embodiment, said multispecific antibody comprises aamino acid T366W mutation in the first CH3 domain of the “knobs chain”and amino acid T366S, L368A, Y407V mutations in the second CH3 domain ofthe “hole chain.” An additional interchain disulfide bridge between theCH3 domains can also be used (Merchant, A. M., et al., Nature Biotech.16 (1998) 677-681) e.g. by introducing an amino acid Y349C mutation intothe CH3 domain of the “hole chain” and an amino acid E356C mutation oran amino acid S354C mutation into the CH3 domain of the “knobs chain.”

In one preferred embodiment, said multispecific antibody (whichcomprises a CH3 domain in each heavy chain) comprises amino acid S354C,T366W mutations in one of the two CH3 domains and amino acid Y349C,T366S, L368A, Y407V mutations in the other of the two CH3 domains (theadditional amino acid S354C mutation in one CH3 domain and theadditional amino acid Y349C mutation in the other CH3 domain forming aninterchain disulfide bridge) (numbering according to Kabat).

Other techniques for CH3-modifications to force the heterodimerizationare contemplated as alternatives of the invention and described e.g. inWO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901,WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment the heterodimerization approach described in EP 1 870459A1, can be used alternatively. This approach is based on theintroduction of substitutions/mutations of charged amino acids with theopposite charge at specific amino acid positions of the CH3/CH3 domaininterface between both heavy chains. One preferred embodiment for saidmultispecific antibody are amino acid R409D; K370E mutations in thefirst CH3 domain of the multispecific antibody, and amino acid D399K,E357K mutations in the second CH3 domain of the multispecific antibody(numbering according to Kabat).

In another embodiment said multispecific antibody comprises an aminoacid T366W mutation in the CH3 domain of the “knobs chain” and aminoacid T366S, L368A, Y407V mutations in the CH3 domain of the “hole chain”and additionally, amino acid R409D; K370E mutations in the CH3 domain ofthe “knobs chain” and amino acid D399K; E357K mutations in the CH3domain of the “hole chain.”

In another embodiment said multispecific antibody comprises amino acidS354C, T366W mutations in one of the two CH3 domains, and amino acidY349C, T366S, L368A, Y407V mutations in the other of the two CH3domains, or said multispecific antibody comprises amino acid Y349C,T366W mutations in one of the two CH3 domains, and amino acid S354C,T366S, L368A, Y407V mutations in the other of the two CH3 domains, andadditionally amino acid R409D; K370E mutations in the CH3 domain of the“knobs chain” and amino acid D399K; E357K mutations in the CH3 domain ofthe “hole chain.”

In one embodiment the heterodimerization approach described inWO2013/157953 can be used. In one embodiment a first CH3 domaincomprises amino acid T366K mutation and a second CH3 domain polypeptidecomprises amino acid L351D mutation. In a further embodiment the firstCH3 domain further comprises an amino acid L351K mutation. In a furtherembodiment the second CH3 domain further comprises an amino acidmutation selected from Y349E, Y349D, and L368E (preferably L368E).

In one embodiment the heterodimerization approach described inWO2012/058768 can be used. In one embodiment a first CH3 domaincomprises amino acid L351Y, Y407A mutations and a second CH3 domaincomprises amino acid T366A, K409F mutations. In a further embodiment thesecond CH3 domain comprises a further amino acid mutation at positionT411, D399, 5400, F405, N390, or K392 e.g. selected from a) T411 N, T411R, T411Q, T411 K, T411D, T411E or T411W, b) D399R, D399W, D399Y orD399K, c S400E, 5400D, 5400R, or 5400K F4051, F405M, F405T, F4055, F405Vor F405W N390R, N390K or N390D K392V, K392M, K392R, K392L, K392F orK392E. In a further embodiment a first CH3 domain comprises amino acidL351Y, Y407A mutations and a second CH3 domain comprises amino acidT366V, K409F mutations. In a further embodiment a first CH3 domaincomprises amino acid Y407A mutations and a second CH3 domain comprisesamino acid T366A, K409F mutations. In a further embodiment the secondCH3 domain comprises a further amino acid K392E, T411E, D399R and 5400Rmutations.

In one embodiment the heterodimerization approach described inWO2011/143545 can be used, e.g. with the amino acid modification at aposition selected from the group consisting of 368 and 409.

In one embodiment the heterodimerization approach described inWO2011/090762 which also uses the knobs-into-holes technology describedabove can be used. In one embodiment a first CH3 domain comprises aminoacid T366W mutations and a second CH3 domain comprises amino acid Y407Amutations. In one embodiment a first CH3 domain comprises amino acidT366Y mutations and a second CH3 domain comprises amino acid Y407Tmutations.

In one embodiment the multispecific antibody is of IgG2 isotype and theheterodimerization approach described in WO2010/129304 can be usedalternatively.

In one embodiment the heterodimerization approach described inWO2009/089004 can be used alternatively. In one embodiment a first CH3domain comprises amino acid substitution of K392 or N392 with anegative-charged amino acid (e.g. glutamic acid (E), or aspartic acid(D), preferably K392D or N392D) and a second CH3 domain comprises aminoacid substitution of D399, E356, D356, or E357 with a positive-chargedamino acid (e.g. Lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K and more preferably D399K and E356K. In a furtherembodiment the first CH3 domain further comprises an amino acidsubstitution of K409 or R409 with a negative-charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D. In afurther embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with anegative-charged amino acid (e.g. glutamic acid (E), or aspartic acid(D)).

In one embodiment the heterodimerization approach described in WO2007/147901 can be used. In one embodiment a first CH3 domain comprisesamino acid K253E, D282K, and K322D mutations and a second CH3 domaincomprises amino acid D239K, E240K, and K292D mutations.

In one embodiment the heterodimerization approach described inWO2007/110205 can be used alternatively.

E. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid(s) encoding a dimeric polypeptide as reportedherein is (are) provided. Such nucleic acid may encode an amino acidsequence comprising the first polypeptide and/or an amino acid sequencecomprising the second polypeptide of the dimeric polypeptide. In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the first polypeptide of the dimeric polypeptide and an aminoacid sequence comprising the second polypeptide of the dimericpolypeptide, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the first polypeptide of thedimeric polypeptide and a second vector comprising a nucleic acid thatencodes an amino acid sequence comprising the second polypeptide of thedimeric polypeptide. In one embodiment, the host cell is eukaryotic,e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,Sp20 cell). In one embodiment, a method of making a dimeric polypeptideas reported herein is provided, wherein the method comprises culturing ahost cell comprising a nucleic acid encoding the dimeric polypeptide, asprovided above, under conditions suitable for expression of the dimericpolypeptide, and optionally recovering the antibody from the host cell(or host cell culture medium).

For recombinant production of a dimeric polypeptide as reported herein,nucleic acid encoding a dimeric polypeptide, e.g., as described above,is isolated and inserted into one or more vectors for further cloningand/or expression in a host cell. Such nucleic acid may be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the variant Fc-region polypeptide(s) and the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of dimericpolypeptide-encoding vectors include prokaryotic or eukaryotic cellsdescribed herein. For example, dimeric polypeptides may be produced inbacteria, in particular when glycosylation and Fc effector function arenot needed. For expression of antibody fragments and polypeptides inbacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523.(See also Charlton, K. A., In: Methods in Molecular Biology, Vol. 248,Lo, B. K. C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254,describing expression of antibody fragments in E. coli). Afterexpression, the dimeric polypeptide may be isolated from the bacterialcell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for dimericpolypeptide-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized” resulting in the productionof a dimeric polypeptide with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of a glycosylated dimericpolypeptide are also derived from multicellular organisms (invertebratesand vertebrates). Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains have been identified whichmay be used in conjunction with insect cells, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (HEK293 or 293cells as described, e.g., in Graham, F. L., et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P., et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR″CHO cells (Urlaub, G., et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

F. Combination Treatment

In certain embodiments the dimeric polypeptide as reported herein orpharmaceutical formulation as reported herein is administered alone(without an additional therapeutic agent) for the treatment of one ormore ocular vascular diseases described herein.

In other embodiments the dimeric polypeptide antibody or pharmaceuticalformulation as reported herein is administered in combination with oneor more additional therapeutic agents or methods for the treatment ofone or more vascular eye diseases described herein.

In other embodiments, the dimeric polypeptide or pharmaceuticalformulation as reported herein is formulated in combination with one ormore additional therapeutic agents and administered for the treatment ofone or more vascular eye diseases described herein.

In certain embodiments, the combination treatments provided hereininclude that the dimeric polypeptide or pharmaceutical formulation asreported herein is administered sequentially with one or more additionaltherapeutic agents for the treatment of one or more ocular vasculardiseases described herein.

The additional therapeutic agents include, but are not limited to,Tryptophanyl-tRNA synthetase (TrpRS), EyeOOl (anti-VEGF PEGylatedaptamer), squalamine, RETAANE™ (anecortave acetate for depot suspension;Alcon, Inc.), Combretastatin A4 Prodrug (CA4P), MACUGEN™, MIFEPREX™(mifepristone-ru486), subtenon triamcinolone acetonide, intravitrealcrystalline triamcinolone acetonide, Prinomastat (AG3340—syntheticmatrix metalloproteinase inhibitor, Pfizer), fluocinolone acetonide(including fluocinolone intraocular implant, Bausch & Lomb/ControlDelivery Systems), VEGFR inhibitors (Sugen), VEGF-Trap(Regeneron/Aventis), VEGF receptor tyrosine kinase inhibitors such as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787) and SU1 1248 (sunitinib), linomide, andinhibitors of integrin vβ3 function and angiostatin.

Other pharmaceutical therapies that can be used in combination with thedimeric polypeptide or pharmaceutical formulation as reported herein,including, but are not limited to, VISUDYNE™ with use of a non-thermallaser, PKC 412, Endovion (NeuroSearch A/S), neurotrophic factors,including by way of example Glial Derived Neurotrophic Factor andCiliary Neurotrophic Factor, diatazem, dorzolamide, Phototrop,9-cis-retinal eye medication (including Echo Therapy) includingphospholine iodide or echothiophate or carbonic anhydrase inhibitors,AE-941 (AEterna Laboratories, Inc.), Sirna-027 (Sima Therapeutics,Inc.), pegaptanib (NeXstar Pharmaceuticals/Gilead Sciences),neurotrophins (including, by way of example only, NT-4/5, Genentech),Candy (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals),integrin antagonists (including those from Jerini AG and AbbottLaboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.),thalidomide (as used, for example, by EntreMed, Inc.), cardiotrophin-1(Genentech), 2-methoxyestradiol (Allergan/Oculex), DL-8234 (TorayIndustries), NTC-200 (Neurotech), tetrathiomolybdate (University ofMichigan), LYN-002 (Lynkeus Biotech), microalgal compound(Aquasearch/Albany, Mera Pharmaceuticals), D-9120 (Celltech Group plc.),ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme/Celtrix), tyrosinekinase inhibitors (Allergan, SUGEN, Pfizer), NX-278-L (NeXstarPharmaceuticals/Gilead Sciences), Opt-24 (OPTIS France SA), retinal cellganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazolederivatives (Texas A&M University System), KP-102 (KrenitskyPharmaceuticals), cyclosporin A, Timited retinal translocation,photodynamic therapy, (including, by way of example only,receptor-targeted PDT, Bristol-Myers Squibb, Co.; porfimer sodium forinjection with PDT; verteporfin, QLT Inc.; rostaporfin with PDT,Miravent Medical Technologies; talaporfin sodium with PDT, NipponPetroleum; motexafin lutetium, Pharmacyclics, Inc.), antisenseoligonucleotides (including, by way of example, products tested byNovagali Pharma SA and ISIS-13650, Isis Pharmaceuticals), laserphotocoagulation, drusen lasering, macular hole surgery, maculartranslocation surgery, implantable miniature telescopes, Phi-MotionAngiography (also known as Micro-Laser Therapy and Feeder VesselTreatment), Proton Beam Therapy, microstimulation therapy, RetinalDetachment and Vitreous Surgery, Scleral Buckle, Submacular Surgery,Transpupillary Thermotherapy, Photosystem I therapy, use of RNAinterference (RNAi), extracorporeal rheopheresis (also known as membranedifferential filtration and Rheotherapy), microchip implantation, stemcell therapy, gene replacement therapy, ribozyme gene therapy (includinggene therapy for hypoxia response element, Oxford Biomedica; Lentipak,Genetix; PDEF gene therapy, GenVec), photoreceptor/retinal cellstransplantation (including transplantable retinal epithelial cells,Diacrin, Inc.; retinal cell transplant, Cell Genesys, Inc.), andacupuncture.

Any anti-angiogenic agent can be used in combination with the dimericpolypeptide or pharmaceutical formulation as reported herein, including,but not limited to, those listed by Carmeliet and Jain (Nature 407(2000) 249-257). In certain embodiments, the anti-angiogenic agent isanother VEGF antagonist or a VEGF receptor antagonist such as VEGFvariants, soluble VEGF receptor fragments, aptamers capable of blockingVEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecular weightinhibitors of VEGFR tyrosine kinases and any combinations thereof andthese include anti-VEGF aptamers (e.g. Pegaptanib), soluble recombinantdecoy receptors (e.g. VEGF Trap). In certain embodiments, theanti-angiogenic agent is include corticosteroids, angiostatic steroids,anecortave acetate, angiostatin, endostatin, small interfering RNA'sdecreasing expression of VEGFR or VEGF ligand, post-VEGFR blockade withtyrosine kinase inhibitors, MMP inhibitors, IGFBP3, SDF-1 blockers,PEDF, gamma-secretase, Delta-like ligand 4, integrin antagonists, HIF-1alpha blockade, protein kinase CK2 blockade, and inhibition of stem cell(i.e. endothelial progenitor cell) homing to the site ofneovascularization using vascular endothelial cadherin (CD-144) andstromal derived factor (SDF)-I antibodies. Small molecule RTK inhibitorstargeting VEGF receptors including PTK787 can also be used. Agents thathave activity against neovascularization that are not necessarilyanti-VEGF compounds can also be used and include anti-inflammatorydrugs, m-Tor inhibitors, rapamycin, everolismus, temsirolismus,cyclospohne, anti-TNF agents, anti-complement agents, and non-steroidalanti-inflammatory agents. Agents that are neuroprotective and canpotentially reduce the progression of dry macular degeneration can alsobe used, such as the class of drugs called the “neurosteroids.” Theseinclude drugs such as dehydroepiandrosterone (DHEA) (Brand names:Prastera® and Fidelin®), dehydroepiandrosterone sulfate, andpregnenolone sulfate. Any AMD (age-related macular degeneration)therapeutic agent can be used in combination with the dimericpolypeptide or pharmaceutical formulation as reported herein, includingbut not limited to verteporfin in combination with PDT, pegaptanibsodium, zinc, or an antioxidant(s), alone or in any combination.

G. Pharmaceutical Formulations

Pharmaceutical formulations of a dimeric polypeptide as reported hereinare prepared by mixing such dimeric polypeptide having the desireddegree of purity with one or more optional pharmaceutically acceptablecarriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.(ed.) (1980)), in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyl dimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rhuPH20, are described in US 2005/0260186 and US2006/0104968. In one aspect, a sHASEGP is combined with one or moreadditional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

H. Therapeutic Methods and Compositions

Any of the dimeric polypeptides as reported herein may be used intherapeutic methods.

In one aspect, a dimeric polypeptide as reported herein for use as amedicament is provided. In further aspects, a dimeric polypeptide foruse in treating ocular vascular diseases is provided. In certainembodiments, a dimeric polypeptide for use in a method of treatment isprovided. In certain embodiments, the invention provides a dimericpolypeptide for use in a method of treating an individual having anocular vascular disease comprising administering to the individual aneffective amount of the dimeric polypeptide as reported herein. In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, e.g., as described above in section D. In further embodiments,the invention provides a dimeric polypeptide for use in inhibitingangiogenesis in the eye. In certain embodiments, the invention providesa dimeric polypeptide for use in a method of inhibiting angiogenesis inan individual comprising administering to the individual an effectiveamount of the dimeric polypeptide to inhibit angiogenesis. An“individual” according to any of the above embodiments is in onepreferred embodiment a human.

In a further aspect, the invention provides for the use of a dimericpolypeptide in the manufacture or preparation of a medicament. In oneembodiment, the medicament is for treatment of an ocular vasculardisease. In a further embodiment, the medicament is for use in a methodof treating an ocular vascular disease comprising administering to anindividual having an ocular vascular disease an effective amount of themedicament. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described above. In a furtherembodiment, the medicament is for inhibiting angiogenesis. In a furtherembodiment, the medicament is for use in a method of inhibitingangiogenesis in an individual comprising administering to the individualan amount effective of the medicament to inhibit angiogenesis. An“individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating avascular eye disease. In one embodiment, the method comprisesadministering to an individual having such a vascular eye disease aneffective amount of a dimeric polypeptide as reported herein. In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, as described below. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for inhibitingangiogenesis in the eye in an individual. In one embodiment, the methodcomprises administering to the individual an effective amount of adimeric polypeptide as reported herein to inhibit angiogenesis. In oneembodiment, an “individual” is a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the dimeric polypeptides as reported herein, e.g., foruse in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the dimeric polypeptides asreported herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the dimericpolypeptides as reported herein and at least one additional therapeuticagent, e.g., as described below.

A dimeric polypeptide as reported herein can be used either alone or incombination with other agents in a therapy. For instance, a dimericpolypeptide as reported herein may be co-administered with at least oneadditional therapeutic agent

A dimeric polypeptide as reported herein (and any additional therapeuticagent) can be administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Dimeric polypeptides as reported herein would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thedimeric polypeptide need not be, but is optionally formulated with oneor more agents currently used to prevent or treat the disorder inquestion. The effective amount of such other agents depends on theamount of dimeric polypeptide present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of adimeric polypeptide as reported herein (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of dimericpolypeptide, the severity and course of the disease, whether the dimericpolypeptide is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to thedimeric polypeptide, and the discretion of the attending physician. Thedimeric polypeptide is suitably administered to the patient at one timeor over a series of treatments. Depending on the type and severity ofthe disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg) ofdimeric polypeptide can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the dimeric polypeptide would be in therange from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more dosesof about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combinationthereof) may be administered to the patient. Such doses may beadministered intermittently, e.g. every week or every three weeks (e.g.such that the patient receives from about two to about twenty, or e.g.about six doses of the dimeric polypeptide). An initial higher loadingdose, followed by one or more lower doses may be administered. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

III. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself, or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a dimeric polypeptide as reported herein. The label orpackage insert indicates that the composition is used for treating thecondition of choice. Moreover, the article of manufacture may comprise(a) a first container with a composition contained therein, wherein thecomposition comprises a dimeric polypeptide as reported herein; and (b)a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate as reported herein in place of or in additionto a dimeric polypeptide as reported herein.

IV. Specific Embodiments

-   1. A dimeric polypeptide comprising    -   a first polypeptide and a second polypeptide each comprising in        N-terminal to C-terminal direction at least a portion of an        immunoglobulin hinge region, which comprises one or more        cysteine residues, an immunoglobulin CH2-domain and an        immunoglobulin CH3-domain,    -   wherein        -   i) the first and the second polypeptide comprise the            mutations H310A, H433A and Y436A, or        -   ii) the first and the second polypeptide comprise the            mutations L251D, L314D and L432D, or        -   iii) the first and the second polypeptide comprise the            mutations L251S, L314S and L432S, or        -   iv) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations H310A, H433A and Y436A, or        -   v) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251D, L314D and L432D, or        -   vi) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251S, L314S and L432S.-   2. The dimeric polypeptide according to item 1, characterized in    that the dimeric polypeptide does not specifically bind to the human    FcRn and does specifically bind to Staphylococcal protein A.-   3. The dimeric polypeptide according to any one of items 1 to 2,    characterized in that the dimeric polypeptide is a homodimeric    polypeptide.-   4. The dimeric polypeptide according to any one of items 1 to 2,    characterized in that the dimeric polypeptide is a heterodimeric    polypeptide.-   5. The dimeric polypeptide according to any one of items 1 to 4,    characterized in that i) the first polypeptide further comprises the    mutations Y349C, T366S, L368A and Y407V and the second polypeptide    comprises the mutations S354C and T366W, or ii) the first    polypeptide further comprises the mutations S354C, T366S, L368A and    Y407V and the second polypeptide comprises the mutations Y349C and    T366W.-   6. The dimeric polypeptide according to any one of items 1 to 5,    characterized in that the immunoglobulin hinge region, the    immunoglobulin CH2-domain and the immunoglobulin CH3-domain are of    the human IgG1 subclass.-   7. The dimeric polypeptide according to any one of items 1 to 6,    characterized in that the first polypeptide and the second    polypeptide further comprise the mutations L234A and L235A.-   8. The dimeric polypeptide according to any one of items 1 to 5,    characterized in that the immunoglobulin hinge region, the    immunoglobulin CH2-domain and the immunoglobulin CH3-domain are of    the human IgG2 subclass optionally with the mutations V234A, G237A,    P238S, H268A, V309L, A330S and P331S.-   9. The dimeric polypeptide according to any one of items 1 to 5,    characterized in that the immunoglobulin hinge region, the    immunoglobulin CH2-domain and the immunoglobulin CH3-domain are of    the human IgG4 subclass.-   10. The dimeric polypeptide according to any one of items 1 to 5 and    9, characterized in that the first polypeptide and the second    polypeptide further comprise the mutations S228P and L235E.-   11. The dimeric polypeptide according to any one of items 1 to 10,    characterized in that the first polypeptide and the second    polypeptide further comprise the mutation P329G.-   12. The dimeric polypeptide according to any one of items 1 to 11,    characterized in that the dimeric polypeptide is an Fc-region fusion    polypeptide.-   13. The dimeric polypeptide according to any one of items 1 to 11,    characterized in that the dimeric polypeptide is an (full length)    antibody.-   14. The dimeric polypeptide according to any one of items 1 to 11    and 13, characterized in that the (full length) antibody is a    monospecific antibody.-   15. The dimeric polypeptide according to any one of items 1 to 11    and 13 to 14, characterized in that the monospecific antibody is a    monovalent monospecific antibody.-   16. The dimeric polypeptide according to any one of items 1 to 11    and 13 to 15, characterized in that the monospecific antibody is a    bivalent monospecific antibody.-   17. The dimeric polypeptide according to any one of items 1 to 11    and 13, characterized in that the (full length) antibody is a    bispecific antibody.-   18. The dimeric polypeptide according to any one of items 1 to 11    and 13 and 17, characterized in that the bispecific antibody is a    bivalent bispecific antibody.-   19. The dimeric polypeptide according to any one of items 1 to 11    and 13 and 17 to 18, characterized in that the bispecific antibody    is a tetravalent bispecific antibody.-   20. The dimeric polypeptide according to any one of items 1 to 11    and 13, characterized in that the (full length) antibody is a    trispecific antibody.-   21. The dimeric polypeptide according to any one of items 1 to 11    and 13 and 20, characterized in that the trispecific antibody is a    trivalent trispecific antibody.-   22. The dimeric polypeptide according to any one of items 1 to 11    and 13 and 20 to 21, characterized in that the trispecific antibody    is a tetravalent trispecific antibody.-   23. A dimeric polypeptide comprising    -   a first polypeptide and a second polypeptide each comprising in        N-terminal to C-terminal direction at least a portion of an        immunoglobulin hinge region, which comprises one or more        cysteine residues, an immunoglobulin CH2-domain and an        immunoglobulin CH3-domain,    -   wherein the first, the second or the first and the second        polypeptide comprise the mutation Y436A (numbering according to        the EU index).-   24. The dimeric polypeptide according to item 23, characterized in    that the first and the second polypeptide comprise the mutation    Y436A.-   25. The dimeric polypeptide according to any one of items 23 to 24,    characterized in that the dimeric polypeptide does not specifically    bind to the human FcRn and does specifically bind to Staphylococcal    protein A.-   26. The dimeric polypeptide according to any one of items 23 to 25,    characterized in that the dimeric polypeptide is a homodimeric    polypeptide.-   27. The dimeric polypeptide according to any one of items 23 to 25,    characterized in that the dimeric polypeptide is a heterodimeric    polypeptide.-   28. The dimeric polypeptide according to any one of items 23 to 27,    characterized in that    -   a) the first polypeptide further comprises the mutations Y349C,        T366S, L368A and Y407V and the second polypeptide comprises the        mutations S354C and T366W,        -   or        -   the first polypeptide further comprises the mutations S354C,            T366S, L368A and Y407V and the second polypeptide comprises            the mutations Y349C and T366W, and/or    -   b) i) the first and the second polypeptide comprise the        mutations H310A, H433A and Y436A, or        -   ii) the first and the second polypeptide comprise the            mutations L251D, L314D and L432D, or        -   iii) the first and the second polypeptide comprise the            mutations L251S, L314S and L432S, or        -   iv) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations H310A, H433A and Y436A, or        -   v) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251D, L314D and L432D, or        -   vi) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251S, L314S and L432S.-   29. The dimeric polypeptide according to any one of items 23 to 28,    characterized in that the immunoglobulin hinge region, the    immunoglobulin CH2-domain and the immunoglobulin CH3-domain are of    the human IgG1 subclass.-   30. The dimeric polypeptide according to any one of items 23 to 29,    characterized in that the first polypeptide and the second    polypeptide further comprise the mutations L234A and L235A.-   31. The dimeric polypeptide according to any one of items 23 to 28,    characterized in that the immunoglobulin hinge region, the    immunoglobulin CH2-domain and the immunoglobulin CH3-domain are of    the human IgG2 subclass optionally with the mutations V234A, G237A,    P238S, H268A, V309L, A330S and P331S.-   32. The dimeric polypeptide according to any one of items 23 to 28,    characterized in that the immunoglobulin hinge region, the    immunoglobulin CH2-domain and the immunoglobulin CH3-domain are of    the human IgG4 subclass.-   33. The dimeric polypeptide according to any one of items 23 to 28    and 32, characterized in that the first polypeptide and the second    polypeptide further comprise the mutations S228P and L235E.-   34. The dimeric polypeptide according to any one of items 23 to 33,    characterized in that the first polypeptide and the second    polypeptide further comprise the mutation P329G.-   35. The dimeric polypeptide according to any one of items 23 to 34,    characterized in that the dimeric polypeptide is an Fc-region fusion    polypeptide.-   36. The dimeric polypeptide according to any one of items 23 to 34,    characterized in that the dimeric polypeptide is an (full length)    antibody.-   37. The dimeric polypeptide according to any one of items 23 to 34    and 36, characterized in that the (full length) antibody is a    monospecific antibody.-   38. The dimeric polypeptide according to any one of items 23 to 34    and 36 to 37, characterized in that the monospecific antibody is a    monovalent monospecific antibody.-   39. The dimeric polypeptide according to any one of items 23 to 34    and 36 to 38, characterized in that the monospecific antibody is a    bivalent monospecific antibody.-   40. The dimeric polypeptide according to any one of items 23 to 34    and 36, characterized in that the (full length) antibody is a    bispecific antibody.-   41. The dimeric polypeptide according to any one of items 23 to 34    and 36 and 40, characterized in that the bispecific antibody is a    bivalent bispecific antibody.-   42. The dimeric polypeptide according to any one of items 23 to 34    and 36 and 40 to 41, characterized in that the bispecific antibody    is a tetravalent bispecific antibody.-   43. The dimeric polypeptide according to any one of items 23 to 34    and 36, characterized in that the (full length) antibody is a    trispecific antibody.-   44. The dimeric polypeptide according to any one of items 23 to 34    and 36 and 43, characterized in that the trispecific antibody is a    trivalent trispecific antibody.-   45. The dimeric polypeptide according to any one of items 23 to 34    and 36 and 43 to 44, characterized in that the trispecific antibody    is a tetravalent trispecific antibody.-   46. A dimeric polypeptide comprising    -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        CH1-domain of the subclass IgG1, an immunoglobulin hinge region        of the subclass IgG1, an immunoglobulin CH2-domain of the        subclass IgG1 and an immunoglobulin CH3-domain of the subclass        IgG1,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG1, an        immunoglobulin hinge region of the subclass IgG1, an        immunoglobulin CH2-domain of the subclass IgG1 and an        immunoglobulin CH3-domain of the subclass IgG1,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and a light chain        constant domain,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen,    -   wherein the second heavy chain variable domain and the second        light chain variable domain form a second binding site that        specifically binds to a second antigen,    -   wherein i) the first polypeptide comprises the mutations Y349C,        T366S, L368A and Y407V and the second polypeptide comprises the        mutations S354C and T366W, or ii) the first polypeptide        comprises the mutations S354C, T366S, L368A and Y407V and the        second polypeptide comprises the mutations Y349C and T366W,    -   wherein the first and the second polypeptide further comprise        the mutations L234A, L235A and P329G, and    -   wherein        -   i) the first and the second polypeptide comprise the            mutations H310A, H433A and Y436A, or        -   ii) the first and the second polypeptide comprise the            mutations L251D, L314D and L432D, or        -   iii) the first and the second polypeptide comprise the            mutations L251S, L314S and L432S, or        -   iv) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations H310A, H433A and Y436A, or        -   v) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251D, L314D and L432D, or        -   vi) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251S, L314S and L432S.-   47. A dimeric polypeptide comprising    -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        light chain constant domain, an immunoglobulin hinge region of        the subclass IgG1, an immunoglobulin CH2-domain of the subclass        IgG1 and an immunoglobulin CH3-domain of the subclass IgG1,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG1, an        immunoglobulin hinge region of the subclass IgG1, an        immunoglobulin CH2-domain of the subclass IgG1 and an        immunoglobulin CH3-domain of the subclass IgG1,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and an        immunoglobulin CH1-domain of the subclass IgG1,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen,    -   wherein the second heavy chain variable domain and the second        light chain variable domain form a second binding site that        specifically binds to a second antigen,    -   wherein i) the first polypeptide comprises the mutations Y349C,        T366S, L368A and Y407V and the second polypeptide comprises the        mutations S354C and T366W, or ii) the first polypeptide        comprises the mutations S354C, T366S, L368A and Y407V and the        second polypeptide comprises the mutations Y349C and T366W,    -   wherein the first and the second polypeptide further comprise        the mutations L234A, L235A and P329G, and    -   wherein        -   i) the first and the second polypeptide comprise the            mutations H310A, H433A and Y436A, or        -   ii) the first and the second polypeptide comprise the            mutations L251D, L314D and L432D, or        -   iii) the first and the second polypeptide comprise the            mutations L251S, L314S and L432S, or        -   iv) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations H310A, H433A and Y436A, or        -   v) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251D, L314D and L432D, or        -   vi) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251S, L314S and L432S.-   48. A dimeric polypeptide comprising    -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        CH1-domain of the subclass IgG4, an immunoglobulin hinge region        of the subclass IgG4, an immunoglobulin CH2-domain of the        subclass IgG4 and an immunoglobulin CH3-domain of the subclass        IgG4,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG4, an        immunoglobulin hinge region of the subclass IgG4, an        immunoglobulin CH2-domain of the subclass IgG4 and an        immunoglobulin CH3-domain of the subclass IgG4,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and a light chain        constant domain,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen,    -   wherein the second heavy chain variable domain and the second        light chain variable domain form a second binding site that        specifically binds to a second antigen,    -   wherein i) the first polypeptide comprises the mutations Y349C,        T366S, L368A and Y407V and the second polypeptide comprises the        mutations S354C and T366W, or ii) the first polypeptide        comprises the mutations S354C, T366S, L368A and Y407V and the        second polypeptide comprises the mutations Y349C and T366W,    -   wherein the first and the second polypeptide further comprise        the mutations S228P, L235E and P329G, and    -   wherein        -   i) the first and the second polypeptide comprise the            mutations H310A, H433A and Y436A, or        -   ii) the first and the second polypeptide comprise the            mutations L251D, L314D and L432D, or        -   iii) the first and the second polypeptide comprise the            mutations L251S, L314S and L432S, or        -   iv) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations H310A, H433A and Y436A, or        -   v) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251D, L314D and L432D, or        -   vi) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251S, L314S and L432S.-   49. A dimeric polypeptide comprising    -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        light chain constant domain, an immunoglobulin hinge region of        the subclass IgG4, an immunoglobulin CH2-domain of the subclass        IgG4 and an immunoglobulin CH3-domain of the subclass IgG4,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG4, an        immunoglobulin hinge region of the subclass IgG4, an        immunoglobulin CH2-domain of the subclass IgG4 and an        immunoglobulin CH3-domain of the subclass IgG4,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and an        immunoglobulin CH1-domain of the subclass IgG4,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen,    -   wherein the second heavy chain variable domain and the second        light chain variable domain form a second binding site that        specifically binds to a second antigen,    -   wherein i) the first polypeptide comprises the mutations Y349C,        T366S, L368A and Y407V and the second polypeptide comprises the        mutations S354C and T366W, or ii) the first polypeptide        comprises the mutations S354C, T366S, L368A and Y407V and the        second polypeptide comprises the mutations Y349C and T366W,    -   wherein the first and the second polypeptide further comprise        the mutations S228P, L235E and P329G, and    -   wherein        -   i) the first and the second polypeptide comprise the            mutations H310A, H433A and Y436A, or        -   ii) the first and the second polypeptide comprise the            mutations L251D, L314D and L432D, or        -   iii) the first and the second polypeptide comprise the            mutations L251S, L314S and L432S, or        -   iv) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations H310A, H433A and Y436A, or        -   v) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251D, L314D and L432D, or        -   vi) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251S, L314S and L432S.-   50. A dimeric polypeptide comprising    -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        CH1-domain of the subclass IgG1, an immunoglobulin hinge region        of the subclass IgG1, an immunoglobulin CH2-domain of the        subclass IgG1, an immunoglobulin CH3-domain of the subclass        IgG1, a peptidic linker and a first scFv,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG1, an        immunoglobulin hinge region of the subclass IgG1, an        immunoglobulin CH2-domain of the subclass IgG1, an        immunoglobulin CH3-domain of the subclass IgG1, a peptidic        linker and a second scFv,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and a light chain        constant domain,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen, the second heavy chain        variable domain and the second light chain variable domain form        a second binding site that specifically binds to a first        antigen, the first and the second scFv specifically bind to a        second antigen,    -   wherein i) the first polypeptide comprises the mutations Y349C,        T366S, L368A and Y407V and the second polypeptide comprises the        mutations S354C and T366W, or ii) the first polypeptide        comprises the mutations S354C, T366S, L368A and Y407V and the        second polypeptide comprises the mutations Y349C and T366W,    -   wherein the first and the second polypeptide further comprise        the mutations L234A, L235A and P329G, and    -   wherein        -   i) the first and the second polypeptide comprise the            mutations H310A, H433A and Y436A, or        -   ii) the first and the second polypeptide comprise the            mutations L251D, L314D and L432D, or        -   iii) the first and the second polypeptide comprise the            mutations L251S, L314S and L432S, or        -   iv) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations H310A, H433A and Y436A, or        -   v) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251D, L314D and L432D, or        -   vi) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251S, L314S and L432S.-   51. A dimeric polypeptide comprising    -   a first polypeptide comprising in N-terminal to C-terminal        direction a first heavy chain variable domain, an immunoglobulin        light chain constant domain, an immunoglobulin hinge region of        the subclass IgG1, an immunoglobulin CH2-domain of the subclass        IgG1, an immunoglobulin CH3-domain of the subclass IgG1, a        peptidic linker and a first scFv,    -   a second polypeptide comprising in N-terminal to C-terminal        direction a second heavy chain variable domain, an        immunoglobulin CH1-domain of the subclass IgG1, an        immunoglobulin hinge region of the subclass IgG1, an        immunoglobulin CH2-domain of the subclass IgG1, an        immunoglobulin CH3-domain of the subclass IgG1, a peptidic        linker and a second scFv,    -   a third polypeptide comprising in N-terminal to C-terminal        direction a first light chain variable domain and an        immunoglobulin CH1-domain of the subclass IgG1,    -   a fourth polypeptide comprising in N-terminal to C-terminal        direction a second light chain variable domain and a light chain        constant domain,    -   wherein the first heavy chain variable domain and the first        light chain variable domain form a first binding site that        specifically binds to a first antigen, the second heavy chain        variable domain and the second light chain variable domain form        a second binding site that specifically binds to a first        antigen, and the first and the second scFv specifically bind to        a second antigen,    -   wherein i) the first polypeptide comprises the mutations Y349C,        T366S, L368A and Y407V and the second polypeptide comprises the        mutations S354C and T366W, or ii) the first polypeptide        comprises the mutations S354C, T366S, L368A and Y407V and the        second polypeptide comprises the mutations Y349C and T366W,    -   wherein the first and the second polypeptide further comprise        the mutations L234A, L235A and P329G, and    -   wherein        -   i) the first and the second polypeptide comprise the            mutations H310A, H433A and Y436A, or        -   ii) the first and the second polypeptide comprise the            mutations L251D, L314D and L432D, or        -   iii) the first and the second polypeptide comprise the            mutations L251S, L314S and L432S, or        -   iv) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations H310A, H433A and Y436A, or        -   v) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251D, L314D and L432D, or        -   vi) the first polypeptide comprises the mutations I253A,            H310A and H435A and the second polypeptide comprises the            mutations L251S, L314S and L432S.-   52. A method for producing a dimeric polypeptide according to any    one of items 1 to 51 comprising the following steps:    -   a) cultivating a mammalian cell comprising one or more nucleic        acids encoding the dimeric polypeptide according to any one of        items 1 to 51,    -   b) recovering the dimeric polypeptide from the cultivation        medium, and    -   c) purifying the dimeric polypeptide with a protein A affinity        chromatography.-   53. Use of the mutation Y436A for increasing the binding of a    dimeric polypeptide to protein A.-   54. Use of the mutations H310A, H433A and Y436A for separating    heterodimeric polypeptides from homodimeric polypeptides.-   55. Use of the mutations L251D, L314D, L432D, or the mutations    L251S, L314S, L432S for separating heterodimeric polypeptides from    homodimeric polypeptides.-   56. Use of the mutations I253A, H310A and H435A in a first    polypeptide in combination with the mutations H310A, H433A and Y436A    in a second polypeptide for separating heterodimeric polypeptides    comprising the first and the second polypeptide from homodimeric    polypeptides.-   57. Use of the mutations I253A, H310A and H435A in a first    polypeptide in combination with the mutations L251D, L314D, L432D or    the mutations L251S, L314S, L432S in a second polypeptide for    separating heterodimeric polypeptides comprising the first and the    second polypeptide from homodimeric polypeptides.-   58. The use according to any one of items 53 to 57, characterized in    that i) the first polypeptide further comprises the mutations Y349C,    T366S, L368A and Y407V and the second polypeptide further comprises    the mutations S354C and T366W, or ii) the first polypeptide    comprises the mutations S354C, T366S, L368A and Y407V and the second    polypeptide comprises the mutations Y349C and T366W.-   59. A method of treatment of a patient suffering from ocular    vascular diseases by administering a dimeric polypeptide according    to any one of items 1 to 51 to a patient in the need of such    treatment.-   60. A dimeric polypeptide according to any one of items 1 to 51 for    intravitreal application.-   61. A dimeric polypeptide according to any one of items 1 to 51 for    the treatment of vascular eye diseases.-   62. A pharmaceutical formulation comprising a dimeric polypeptide    according to any one of items 1 to 51 and optionally a    pharmaceutically acceptable carrier.-   63. Use of a dimeric polypeptide according to any one of items 1 to    51 for the transport of a soluble receptor ligand from the eye over    the blood-ocular-barrier into the blood circulation.-   64. Use of a dimeric polypeptide according to any one of items 1 to    51 for the removal of one or more soluble receptor ligands from the    eye.-   65. Use of a dimeric polypeptide according to any one of items 1 to    51 for the treatment of eye diseases, especially of ocular vascular    diseases.-   66. Use of a dimeric polypeptide according to any one of items 1 to    51 for the transport of one or more soluble receptor ligands from    the intravitreal space to the blood circulation.-   67. A dimeric polypeptide according to any one of items 1 to 51 for    use in treating an eye disease.-   68. A dimeric polypeptide according to any one of items 1 to 51 for    use in the transport of a soluble receptor ligand from the eye over    the blood-ocular-barrier into the blood circulation.-   69. A dimeric polypeptide according to any one of items 1 to 51 for    use in the removal of one or more soluble receptor ligands from the    eye.-   70. A dimeric polypeptide according to any one of items 1 to 51 for    use in treating eye diseases, especially ocular vascular diseases.-   71. A dimeric polypeptide according to any one of items 1 to 51 for    use in the transport of one or more soluble receptor ligands from    the intravitreal space to the blood circulation.-   72. A method of treating an individual having an ocular vascular    disease comprising administering to the individual an effective    amount of a dimeric polypeptide according to any one of items 1 to    51.-   73. A method for transporting a soluble receptor ligand from the eye    over the blood-ocular-barrier into the blood circulation in an    individual comprising administering to the individual an effective    amount of a dimeric polypeptide according to any one of items 1 to    51 to transport a soluble receptor ligand from the eye over the    blood-ocular-barrier into the blood circulation.-   74. A method the removal of one or more soluble receptor ligands    from the eye in an individual comprising administering to the    individual an effective amount of a dimeric polypeptide according to    any one of items 1 to 51 to remove one or more soluble receptor    ligands from the eye.-   75. A method for the transport of one or more soluble receptor    ligands from the intravitreal space to the blood circulation in an    individual comprising administering to the individual an effective    amount of a dimeric polypeptide according to any one of items 1 to    51 to transport of one or more soluble receptor ligands from the    intravitreal space to the blood circulation.-   76. A method for transporting a soluble receptor ligand from the    intravitreal space or the eye over the blood-ocular-barrier into the    blood circulation in an individual comprising administering to the    individual an effective amount of a dimeric polypeptide according to    any one of items 1 to 51 to transport a soluble receptor ligand from    the eye over the blood-ocular-barrier into the blood circulation.

V. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Methods

Electrospray Ionization Mass Spectrometry (ESI-MS)

Protein aliquots (50 μg) were deglycosylated by adding 0.5 μLN-Glycanase plus (Roche) and sodium phosphate buffer (0.1 M, pH 7.1) toobtain a final sample volume of 115 μL. The mixture was incubated at 37°C. for 18 h. Afterwards for reduction and denaturing 60 μL 0.5 M TCEP(Pierce) in 4 M guanidine*HCl (Pierce) and 50 μL 8 M guanidine*HCl wereadded. The mixture was incubated at 37° C. for 30 min. Samples weredesalted by size exclusion chromatography (Sepharose G-25, isocratic,40% acetonitrile with 2% formic acid). ESI mass spectra (+ve) wererecorded on a Q-TOF instrument (maXis, Bruker) equipped with a nano ESIsource (TriVersa NanoMate, Advion). MS parameter settings were asfollows: Transfer: Funnel RF, 400 Vpp; ISCID Energy, 0 eV; Multipole RF,400 Vpp; Quadrupole: Ion Energy, 4.0 eV; Low Mass, 600 m/z; Source: DryGas, 8 L/min; Dry Gas Temperature, 160° C.; Collision Cell: CollisionEnergy, 10 eV; Collision RF: 2000 Vpp; Ion Cooler: Ion Cooler RF, 300Vpp; Transfer Time: 120 μs; Pre Puls Storage, 10 μs; scan range m/z 600to 2000. For data evaluation in-house developed software (MassAnalyzer)was used.

FcRn Surface Plasmon Resonance (SPR) Analysis

The binding properties of wild-type antibody and the mutants to FcRnwere analyzed by surface plasmon resonance (SPR) technology using aBIAcore T100 instrument (BIAcore AB, Uppsala, Sweden). This system iswell established for the study of molecular interactions. It allows acontinuous real-time monitoring of ligand/analyte bindings and thus thedetermination of kinetic parameters in various assay settings.SPR-technology is based on the measurement of the refractive index closeto the surface of a gold coated biosensor chip. Changes in therefractive index indicate mass changes on the surface caused by theinteraction of immobilized ligand with analyte injected in solution. Ifmolecules bind to an immobilized ligand on the surface the massincreases, in case of dissociation the mass decreases. In the currentassay, the FcRn receptor was immobilized onto a BIAcore CM5-biosensorchip (GE Healthcare Bioscience, Uppsala, Sweden) via amine coupling to alevel of 400 Response units (RU). The assay was carried out at roomtemperature with PBS, 0.05% Tween20 pH 6.0 (GE Healthcare Bioscience) asrunning and dilution buffer. 200 nM of samples were injected at a flowrate of 50 μL/min at room temperature. Association time was 180 sec.,dissociation phase took 360 sec. Regeneration of the chip surface wasreached by a short injection of HBS-P, pH 8.0. Evaluation of SPR-datawas performed by comparison of the biological response signal height at180 sec. after injection and at 300 sec. after injection. Thecorresponding parameters are the RU max level (180 sec. after injection)and late stability (300 sec. after end of injection).

Protein a Surface Plasmon Resonance (SPR) Analysis

The assay is based on surface plasmon resonance spectroscopy. Protein Ais immobilized onto the surface of a SPR biosensor. By injecting thesample into the flow cells of the SPR spectrometer it forms a complexwith the immobilized protein A resulting in an increasing mass on thesensor chip surface, and therefore to a higher response (as 1 RU isdefined as 1 pg/mm²). Afterwards the sensor chip is regenerated bydissolving the sample-protein A-complex. The gained responses are thenevaluated for the signal high in response units (RU) and thedissociation behavior

Around 3500 response units (RU) of protein A (20 μg/mL) were coupledonto a CM5 chip (GE Healthcare) at pH 4.0 by using the amine couplingkit of GE Healthcare.

The sample and system buffer was HBS-P+ (0.01 M HEPES, 0.15 M NaCl,0.005% Surfactant P20 Sterile-filtered, pH 7.4). Flow cell temperaturewas set to 25° C. and sample compartment temperature to 12° C. Thesystem was primed with running buffer. Then, 5 nM solutions of thesample constructs were injected for 120 seconds with a flow rate of 30μL/min, followed by a 300 seconds dissociation phase. Then the sensorchip surface was regenerated by two 30 seconds long injections ofGlycine-HCl pH 1.5 at a flow rate of 30 μL/min. Each sample was measuredas a triplicate.

Bispecific Antibodies and their Respective Sequences

Description Sequences anti-VEGF/ANG2 CrossMab SEQ ID NO: 34, SEQ ID IgG1with IHH-AAA mutations NO: 35, SEQ ID NO: 36, SEQ ID NO: 37anti-VEGF/ANG2 CrossMab SEQ ID NO: 52, SEQ ID IgG1 wild type (withoutIHH- NO: 53, SEQ ID NO: 54, AAA mutations) SEQ ID NO: 55 anti-VEGF/ANG2CrossMab SEQ ID NO: 38, SEQ ID IgG1 with IHH-AAA mutations NO: 39, SEQID NO: 40, and P329G LALA mutations SEQ ID NO: 41 anti-VEGF/ANG2CrossMab SEQ ID NO: 56, SEQ ID IgG1 with P329G LALA NO: 57, SEQ ID NO:58, mutations only (without IHH- SEQ ID NO: 59 AAA mutations)anti-VEGF/ANG2 CrossMab SEQ ID NO: 42, SEQ ID IgG4 with IHH-AAAmutations NO: 43, SEQ ID NO: 44, and with SPLE mutations SEQ ID NO: 45anti-VEGF/ANG2 OAscFab SEQ ID NO: 46, SEQ ID IgG1 with IHH-AAA mutationsNO: 47, SEQ ID NO: 48 <VEGF-ANG-2> OAscFab SEQ ID NO: 49, SEQ ID IgG4with IHH-AAA mutations NO: 50, SEQ ID NO: 51 and with SPLE mutationsanti-VEGF/ANG2 CrossMab SEQ ID NO: 102, SEQ ID IgG1 with HHY-AAA NO:103, SEQ ID NO: 36, mutations SEQ ID NO: 37 anti-VEGF/ANG2 CrossMab SEQID NO: 104, SEQ ID IgG1 with HHY-AAA NO: 105, SEQ ID NO: 36, mutationsand P329G LALA SEQ ID NO: 37 mutations anti-VEGF/ANG2 CrossMab SEQ IDNO: 106, SEQ ID IgG4 with HHY-AAA NO: 107, SEQ ID NO: 58, mutations andwith SPLE SEQ ID NO: 59 mutations <VEGF-ANG-2> OAscFab SEQ ID NO: 108,SEQ ID IgG1 with HHY-AAA NO: 109, SEQ ID NO: 48 mutations <VEGF-ANG-2>OAscFab SEQ ID NO: 110, SEQ ID IgG4 with HHY-AAA NO: 111, SEQ ID NO: 51mutations and with SPLE mutations

The term “with (the) mutation IHH-AAA” as used herein refers thecombination of the mutations I253A (Ile253Ala), H310A (His310Ala), andH435A (His435Ala) in a constant heavy chain region of IgG1 or IgG4subclass (numbering according to the Kabat EU index numbering system),the term “with (the) mutation HHY-AAA” as used herein refers thecombination of the mutations H310A (His310Ala), H433A (His433Ala) andY436A (Tyr436Ala) in a constant heavy chain region of IgG1 or IgG4subclass (numbering according to the Kabat EU index numbering system),the term “with (the) mutation P329G LALA” as used herein refers to thecombination of the mutations L234A (Leu234Ala), L235A (Leu235Ala) andP329G (Pro329Gly) in a constant heavy chain region of IgG1 subclass(numbering according to the Kabat EU index numbering system), and theterm “with (the) mutation SPLE” as used herein refers to the combinationof the mutations S228P (Ser228Pro) and L235E (Leu235Glu) in a constantheavy chain region of IgG4 subclass (numbering according to the Kabat EUindex numbering system).

Description Sequences <IGF-1R> IgG1 wt SEQ ID NO: 88 SEQ ID NO: 89<IGF-1R> IgG1 with SEQ ID NO: 88 I253A, H310A, H435A SEQ ID NO: 90<IGF-1R> IgG1 with SEQ ID NO: 88 M252Y, S254T, T256E SEQ ID NO: 91<IgF-1R> IgG1 wt, KiH SEQ ID NO: 88 SEQ ID NO: 92 SEQ ID NO: 93 <IgF-1R>IgG1 knob wt, SEQ ID NO: 88 hole I253A, H310A, SEQ ID NO: 94 H435A SEQID NO: 95 <IGF-1R> IgG1 knob wt, SEQ ID NO: 88 hole H310A, H433A, SEQ IDNO: 96 Y436A SEQ ID NO: 97 <IGF-1R> IgG1 knob wt, SEQ ID NO: 88 holeM252Y, S254T, SEQ ID NO: 98 T256E SEQ ID NO: 99 <IGF-1R> IgG1 knob wt,SEQ ID NO: 88 hole L251D, L314D, SEQ ID NO: 100 L432D SEQ ID NO: 101<IGF-1R> IgG1 with SEQ ID NO: 88 H310A, H433A, Y436A SEQ ID NO: 112General

General information regarding the nucleotide sequences of humanimmunoglobulin light and heavy chains is given in: Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Amino acidresidues of antibody chains are numbered and referred to according to EUnumbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63 (1969)78-85; Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991)).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular Cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were ordered according to given specifications atGeneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies expression vectors fortransient expression (e.g. in HEK293-F cells) based either on a cDNAorganization with or without a CMV-Intron A promoter or on a genomicorganization with a CMV promoter were used.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this vector        in E. coli,    -   a ß-lactamase gene which confers ampicillin resistance in E.        coli, and    -   the dihydrofolate reductase gene from Mus musculus as a        selectable marker in eukaryotic cells.

The transcription unit of the antibody gene was composed of thefollowing elements:

-   -   unique restriction site(s) at the 5′ end,    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   in the case of the cDNA organization followed by the Intron A        sequence,    -   a 5′-untranslated region of a human immunoglobulin gene,    -   a nucleic acid encoding an immunoglobulin heavy chain signal        sequence,    -   a nucleic acid encoding the human antibody chain (wild-type or        with domain exchange) either as cDNA or in genomic organization        with the immunoglobulin exon-intron organization,    -   a 3′ non-translated region with a polyadenylation signal        sequence, and    -   unique restriction site(s) at the 3′ end.

The nucleic acids encoding the antibody chains were generated by PCRand/or gene synthesis and assembled by known recombinant methods andtechniques by connection of the according nucleic acid segments e.g.using unique restriction sites in the respective vectors. The subclonednucleic acid sequences were verified by DNA sequencing. For transienttransfections, larger quantities of the vectors were prepared by vectorpreparation from transformed E. coli cultures (Nucleobond AX,Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

The bispecific antibodies were expressed by transient co-transfection ofthe respective expression vectors in HEK29-F cells growing in suspensionas described below.

Example 1

Expression and Purification

Transient Transfections in HEK293-F System

The monospecific and bispecific antibodies were generated by transienttransfection with the respective vectors (e.g. encoding the heavy andmodified heavy chain, as well as the corresponding light and modifiedlight chain) using the HEK293-F system (Invitrogen) according to themanufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growingin suspension either in a shake flask or in a stirred fermenter inserum-free FREESTYLE™ 293 expression medium (Invitrogen) weretransfected with a mix of the respective expression vectors and293FECTIN™ or fectin (Invitrogen). For 2 L shake flask (Corning)HEK293-F cells were seeded at a density of 1*10⁶ cells/mL in 600 mL andincubated at 120 rpm, 8% CO₂. The day after the cells were transfectedat a cell density of approx. 1.5*10⁶ cells/mL with approx. 42 mL mix ofA) 20 mL Opti-MEM (Invitrogen) with 600 μg total vector DNA (1 μg/mL)encoding the heavy or modified heavy chain, respectively and thecorresponding light chain in an equimolar ratio and B) 20 ml Opti-MEMwith 1.2 mL 293 fectin or fectin (2 μL/mL). According to the glucoseconsumption, glucose solution was added during the course of thefermentation. The supernatant containing the secreted antibody washarvested after 5-10 days and antibodies were either directly purifiedfrom the supernatant or the supernatant was frozen and stored.

Purification

Bispecific antibodies were purified from cell culture supernatants byaffinity chromatography using MABSELECTSURE-SEPHAROSE™ (for non-IHH-AAAmutants) (GE Healthcare, Sweden) or KappaSelect-Agarose (for IHH-AAAmutants) (GE Healthcare, Sweden), hydrophobic interaction chromatographyusing butyl-Sepharose (GE Healthcare, Sweden) and Superdex 200 sizeexclusion (GE Healthcare, Sweden) chromatography.

Briefly, sterile filtered cell culture supernatants were captured on aMABSELECTSURE™ resin equilibrated (non-IHH-AAA mutations and wild-typeantibodies) with PBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25mM sodium citrate at pH 3.0. The IHH-AAA mutants were captured on aKappaSelect resin equilibrated with 25 mM Tris, 50 mM NaCl, pH 7.2,washed with equilibration buffer and eluted with 25 mM sodium citrate pH2.9. The eluted antibody fractions were pooled and neutralized with 2 MTris, pH 9.0. The antibody pools were prepared for hydrophobicinteraction chromatography by adding 1.6 M ammonium sulfate solution toa final concentration of 0.8 M ammonium sulfate and the pH adjusted topH 5.0 using acetic acid. After equilibration of the butyl-Sepharoseresin with 35 mM sodium acetate, 0.8 M ammonium sulfate, pH 5.0, theantibodies were applied to the resin, washed with equilibration bufferand eluted with a linear gradient to 35 mM sodium acetate pH 5.0. The(monospecific or bispecific) antibody containing fractions were pooledand further purified by size exclusion chromatography using a Superdex200 26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mMhistidine, 140 mM NaCl, pH 6.0. The (monospecific or bispecific)antibody containing fractions were pooled, concentrated to the requiredconcentration using Vivaspin ultrafiltration devices (Sartorius StedimBiotech S. A., France) and stored at −80° C.

TABLE Yields of bispecific <VEGF-ANG-2> antibodies VEGF/ VEGF/ ANG2-0015ANG2-0016 (without (with VEGF/ANG2- IHH-AAA IHH-AAA 0121 (with HHY-mutation) mutation) AAA mutation) titer supernatant 64 μg/mL, n.a. (2 Lscale) 60.8 μg/mL (2 L = (2 L = 128 mg) 121.60 mg) protein A 118 mg(~70% n.a. 100.5 mg (pool1 + (MabSelectSure) monomer) pool2) KappaSelect n.a. 117 mg (~83% n.a. monomer) Butyl Sepharose 60 mg 57 mg 49 mgSEC 35 mg (>95% 38 mg (>95% 32.4 mg (>95% monomer) monomer) monomer)

Purity and antibody integrity were analyzed after each purification stepby CE-SDS using microfluidic Labchip technology (Caliper Life Science,USA). Five μL of protein solution was prepared for CE-SDS analysis usingthe HT Protein Express Reagent Kit according manufacturer's instructionsand analyzed on Labchip GXII system using a HT Protein Express Chip.Data were analyzed using Labchip GX Software.

TABLE Removal of typical side products by different sequentialpurification steps determined by CE-SDS. VEGF/ANG2-0015 VEGF/ANG2-0016purification % peak area* * analysis: CE-SDS (Caliper Labchip GXII) stepmAb ¾Ab (HC)2 ½Ab (LC)2 LC mAb ¾Ab (HC)2 ½Ab (LC)2 LC MAb Select 55.7 1910.6 9.8 3.5 0.9 — Sure Kappa Select — 63 13.4 3.5 6.1 5.8 7.4ButylSepharose 81.4 1.9 2.3 8.2 3.6 1.8 76.2 1.3 0.7 8.3 7.7 5.8Superdex 200 92.4 1.8 2.6 1.4 0.5 0.5 99 1.1 n.d. n.d. n.d. n.d. SEC

The aggregate content of antibody samples was analyzed byhigh-performance SEC using a Superdex 200 analytical size-exclusioncolumn (GE Healthcare, Sweden) in 2×PBS (20 mM Na₂HPO₄, 2 mM KH₂PO₄, 274mM NaCl and 5.4 mM KCl, pH 7.4) running buffer at 25° C. 25 μg proteinwere injected on the column at a flow rate of 0.75 mL/min and elutedisocratic over 50 minutes.

Analogously, the anti-VEGF/ANG2 antibodies VEGF/ANG2-0012 andVEGF/ANG2-0201 were prepared and purified with the following yields:

VEGF/ANG2-0012 VEGF/ANG2-0201 (with IHH-AAA (without mutation) IHH-AAAmutation) titer //amount — 36 μg/mL/72 mg scale 2.1 L 2 L protein A — 66mg (~95% (MabSelectSure) monomer) KappaSelect 43 mg (~65% monomer) —Butyl Sepharose — 45 mg SEC 14 mg 21 mg (>98% monomer) yieldhydroxylapatite 8.5 mg (>98% monomer) total yield (recovery) 8.5 mg(20%) 21 mg (30%)

Also the anti-VEGF/ANG2 bispecific antibodies anti-VEGF/ANG2 CrossMAbIgG4 with IHH-AAA mutation and with SPLE mutation (SEQ ID NO: 42, SEQ IDNO: 43, SEQ ID NO: 44, SEQ ID NO: 45), anti-VEGF/ANG2 OAscFab IgG1 withIHH-AAA mutation (SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48),anti-VEGF/ANG2 OAscFab IgG4 with IHH-AAA mutation and with SPLE mutation(SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51), anti-VEGF/ANG2 CrossMabIgG1 with HHY-AAA mutation and P329G LALA mutation (SEQ ID NO: 90, SEQID NO: 91, SEQ ID NO: 40, SEQ ID NO: 41), anti-VEGF/ANG2 CrossMab IgG4with HHY-AAA mutation and SPLE mutation (SEQ ID NO: 92, SEQ ID NO: 93,SEQ ID NO: 44, SEQ ID NO: 45), anti-VEGF/ANG2 OAscFab IgG1 with HHY-AAAmutation (SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 48), andanti-VEGF/ANG2 OAscFab IgG4 with HHY-AAA mutation and SPLE mutation (SEQID NO: 96, SEQ ID NO: 97, SEQ ID NO: 51) and also the anti-IGF-1Rmonospecific antibodies anti-IGF-1R wild-type (SEQ ID NO: 88, SEQ ID NO:89), anti-IGF-1R IgG1 with IHH-AAA mutation (SEQ ID NO: 88, SEQ ID NO:90), anti-IGF-1R IgG1 with YTE mutation (SEQ ID NO: 88, SEQ ID NO: 91),anti-IGF-1R IgG1 wild-type with KiH mutation (SEQ ID NO: 88, SEQ ID NO:92, SEQ ID NO: 93), anti-IGF-1R IgG1 with KiH mutation and the IHH-AAAmutation in the hole chain (SEQ ID NO: 88, SEQ ID NO: 94, SEQ ID NO:95), anti-IGF-1R IgG1 with KiH mutation and the HHY-AAA mutation in thehole chain (SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 97), anti-IGF-1RIgG1 with KiH mutation and the YTE mutation (SEQ ID NO: 88, SEQ ID NO:98, SEQ ID NO: 99), anti-IGF-1R IgG1 with KiH mutation and the DDDmutation (SEQ ID NO: 88, SEQ ID NO: 100, SEQ ID NO: 101), andanti-IGF-1R IgG1 with HHY-AAA mutation (SEQ ID NO: 88, SEQ ID NO: 112)can be prepared and purified analogously.

Example 2

Analytics & Developability

Small-Scale DLS-Based Viscosity Measurement.

Viscosity measurement was essentially performed as described in (He, F.et al., Analytical Biochemistry 399 (2009) 141-143). Briefly, samplesare concentrated to various protein concentrations in 200 mM argininesuccinate, pH 5.5, before polystyrene latex beads (300 nm diameter) andPolysorbate 20 (0.02% v/v) are added. Samples are transferred into anoptical 384-well plate by centrifugation through a 0.4 μm filter plateand covered with paraffin oil. The apparent diameter of the latex beadsis determined by dynamic light scattering at 25° C. The viscosity of thesolution can be calculated as η=η0(rh/rh,0) (η: viscosity; η0: viscosityof water; rh: apparent hydrodynamic radius of the latex beads; rh,0:hydrodynamic radius of the latex beads in water).

To allow comparison of various samples at the same concentration,viscosity-concentration data were fitted with the Mooney equation(Equation 1) (Mooney, M., Colloid. Sci., 6 (1951) 162-170; Monkos, K.,Biochem. Biophys. Acta 304 (1997) 1339) and data interpolatedaccordingly.

$\begin{matrix}{\eta = {\eta_{0}{\exp\left( \frac{S\;\Phi}{1 - {K\;\Phi}} \right)}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$(S: hydrodynamic interaction parameter of the protein; K: self-crowdingfactor; Φ: volume fraction of the dissolved protein)

Results are shown in FIG. 2 : VEGF/ANG2-0016 with IHH-AAA mutation inthe Fc-region shows a lower viscosity at all measured temperaturescompared to VEGF/ANG2-0015 without the IHH-AAA mutation in theFc-region.

DLS Aggregation Onset Temperature

Samples are prepared at a concentration of 1 mg/mL in 20 mMhistidine/histidine hydrochloride, 140 mM NaCl, pH 6.0, transferred intoan optical 384-well plate by centrifugation through a 0.4 μm filterplate and covered with paraffin oil. The hydrodynamic radius is measuredrepeatedly by dynamic light scattering while the samples are heated witha rate of 0.05° C./min from 25° C. to 80° C. The aggregation onsettemperature is defined as the temperature at which the hydrodynamicradius starts to increase. Results are shown in FIG. 3 . In FIG. 3 theaggregation of VEGF/ANG2-0015 without the IHH-AAA mutation versusVEGF/ANG2-0016 with IHH-AAA mutation in the Fc-region is shown.VEGF/ANG2-0016 showed an aggregation onset temperature of 61° C. whereasVEGF/ANG2-0015 without the IHH-AAA mutation showed an onset temperatureof 60° C.

DLS Time-Course

Samples are prepared at a concentration of 1 mg/mL in 20 mMhistidine/histidine hydrochloride, 140 mM NaCl, pH 6.0, transferred intoan optical 384-well plate by centrifugation through a 0.4 μm filterplate and covered with paraffin oil. The hydrodynamic radius is measuredrepeatedly by dynamic light scattering while the samples are kept at aconstant temperature of 50° C. for up to 145 hours. In this experiment,aggregation tendencies of the native, unfolded protein at elevatedtemperature would lead to an increase of the average particle diameterover time. This DLS-based method is very sensitive for aggregatesbecause these contribute over-proportionally to the scattered lightintensity. Even after 145 hours at 50° C. (a temperature close to theaggregation-onset temperature, see above), an average particle diameterincrease of only less than 0.5 nm was found for both VEGF/ANG2-0015 andVEGF/ANG2-0016.

Seven Day Storage at 40° C. at 100 mg/mL

Samples are concentrated to a final concentration of 100 mg/mL in 200 mMarginine succinate, pH 5.5, sterile filtered and quiescently stored at40° C. for 7 days. Before and after storage, the content of high and lowmolecular weight species (HMWs and LMWs, respectively) is determined bysize-exclusion chromatography. The difference in HMW and LMW contentbetween the stored sample and a sample measured immediately afterpreparation is reported as “HMW increase” and “LMW increase,”respectively. Results are shown in the Table below and FIG. 4 , whichshow that VEGF/ANG2-0015 (without IHH-AAA mutation) shows a higherreduction of the main peak and a higher HMW increase compared toVEGF/ANG2-0016 (with IHH-AAA mutation). Surprisingly VEGF/ANG2-0016(with IHH-AAA mutation) showed a lower aggregation tendency compared toVEGF/ANG2-0015 (without IHH-AAA mutation).

TABLE Delta Main-, HMW and LMW peaks after 7 d at 40° C. delta_area %(40° C.-(−80° C.)) main Peak HMW LMW VEGF/ANG2-0015 −3.56 2.89 0.67(without IHH-AAA mutation) VEGF/ANG2-0016 −1.74 1.49 0.25 (with IHH-AAAmutation)

The functional analysis of anti-VEGF/ANG2 bispecific antibodies wasassessed by Surface Plasmon Resonance (SPR) using a BIAcore® T100 orT200 instrument (GE Healthcare) at 25° C. The BIAcore® system is wellestablished for the study of molecule interactions. SPR-technology isbased on the measurement of the refractive index close to the surface ofa gold coated biosensor chip. Changes in the refractive index indicatemass changes on the surface caused by the interaction of immobilizedligand with analyte injected in solution. The mass increases ifmolecules bind immobilized ligands on the surface, and vice versa, themass decreases in case of dissociation of the analyte from theimmobilized ligand (reflecting complex dissociation). SPR allows acontinuous real-time monitoring of ligand/analyte binding and thusdetermination of the association rate constant (ka), dissociation rateconstant (kd), and the equilibrium constant (KD).

Example 3

Binding to VEGF, ANG2, FcgammaR and FcRn

VEGF Isoforms Kinetic Affinity Including Assessment ofSpecies-Cross-Reactivity

Around 12,000 resonance units (RU) of the capturing system (10 μg/mLgoat anti human F(ab)′₂; Order Code: 28958325; GE HealthcareBio-Sciences AB, Sweden) were coupled on a CM5 chip (GE HealthcareBR-1005-30) at pH 5.0 by using an amine coupling kit supplied by GEHealthcare. The sample and system buffer was PBS-T (10 mM phosphatebuffered saline including 0.05% Tween20) pH 7.4. The flow cell was setto 25° C.—and the sample block set to 12° C.—and primed with runningbuffer twice. The bispecific antibody was captured by injecting a 50 nMsolution for 30 seconds at a flow of 5 μL/min. Association was measuredby injection of human hVEGF121, mouse mVEGF120 or rat rVEGF164 invarious concentrations in solution for 300 seconds at a flow of 30μL/min starting with 300 nM in 1:3 dilutions. The dissociation phase wasmonitored for up to 1200 seconds and triggered by switching from thesample solution to running buffer. The surface was regenerated by 60seconds washing with a Glycine pH 2.1 solution at a flow rate of 30μL/min. Bulk refractive index differences were corrected by subtractingthe response obtained from a goat anti human F(ab′)₂ surface. Blankinjections are also subtracted (=double referencing). For calculation ofapparent K_(D) and other kinetic parameters, the Langmuir 1:1 model wasused. Results are shown below.

ANG2 Solution Affinity Including Assessment of Species-Cross-Reactivity

Solution affinity measures the affinity of an interaction by determiningthe concentration of free interaction partners in an equilibriummixture. The solution affinity assay involves the mixing of ananti-VEGF/ANG2 antibody, kept at a constant concentration, with a ligand(=ANG2) at varying concentrations. Maximum possible resonance units(e.g. 17,000 resonance units (RU)) of an antibody was immobilized on theCM5 chip (GE Healthcare BR-1005-30) surface at pH 5.0 using an aminecoupling kit supplied by GE Healthcare. The sample and system buffer wasHBS-P pH 7.4. Flow cell was set to 25° C. and sample block to 12° C. andprimed with running buffer twice. To generate a calibration curve,increasing concentrations of ANG2 were injected into a BIAcore flow-cellcontaining the immobilized anti-VEGF/ANG2 antibody. The amount of boundANG2 was determined as resonance units (RU) and plotted against theconcentration. Solutions of each ligand (11 concentrations from 0 to 200nM for the anti-VEGF/ANG2 antibody) were incubated with 10 nM ANG2 andallowed to reach equilibrium at room temperature. Free ANG2concentrations were determined from the calibration curve generatedbefore and after measuring the response of solutions with known amountsof ANG2. A 4-parameter fit was set with XLfit4 (IDBS Software) usingModel 201 using free ANG2 concentration as y-axis and used concentrationof antibody for inhibition as x-axis. The affinity was calculated bydetermining the inflection point of this curve. The surface wasregenerated by one time 30 seconds washing with a 0.85% H₃PO₄ solutionat a flow rate of 30 μL/min. Bulk refractive index differences werecorrected by subtracting the response obtained from a blank-coupledsurface. Results are shown in below.

FcRn Steady State Affinity

For FcRn measurement, a steady state affinity was used to comparebispecific antibodies against each other. Human FcRn was diluted intocoupling buffer (10 μg/mL, Na-Acetate, pH 5.0) and immobilized on aC1-Chip (GE Healthcare BR-1005-35) by targeted immobilization procedureusing a BIAcore wizard to a final response of 200 RU. Flow cell was setto 25° C. and sample block to 12° C. and primed with running buffertwice. The sample and system buffer was PBS-T (10 mM phosphate bufferedsaline including 0.05% Tween20) pH 6.0. To assess different IgGconcentrations for each antibody, a concentration of 62.5 nM, 125 nM,250 nM, and 500 nM was prepared. Flow rate was set to 30 μL/min and thedifferent samples were injected consecutively onto the chip surfacechoosing 180 seconds association time. The surface was regenerated byinjected PBS-T pH 8 for 60 seconds at a flow rate of 30 μL/min. Bulkrefractive index differences were corrected by subtracting the responseobtained from a blank surface. Buffer injections are also subtracted(=double referencing). For calculation of steady state affinity themethod from the BIA-Evaluation software was used. Briefly, the RU valueswere plotted against the analyzed concentrations, yielding adose-response curve. Based on a 2-parametric fit, the upper asymptote iscalculated, allowing the determination of the half-maximal RU value andhence the affinity. Results are shown in FIG. 5 and the Table below.Analogously the affinity to Cynomolgus, mouse and rabbit FcRn can bedetermined.

FcgammaRIIIa Measurement

For FcgammaRIIIa measurement a direct binding assay was used. Around3,000 resonance units (RU) of the capturing system (1 μg/mL Penta-His;Qiagen) were coupled on a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0by using an amine coupling kit supplied by GE Healthcare. The sample andsystem buffer was HBS-P+ pH 7.4. The flow cell was set to 25° C.—andsample block to 12° C.—and primed with running buffer twice. TheFcgammaRIIIa-His-receptor was captured by injecting a 100 nM solutionfor 60 seconds at a flow of 5 μL/min. Binding was measured by injectionof 100 nM of bispecific antibody or monospecific control antibodies(anti-digoxygenin antibody for IgG1 subclass and an IgG4 subclassantibody) for 180 seconds at a flow of 30 μL/min. The surface wasregenerated by 120 seconds washing with Glycine pH 2.5 solution at aflow rate of 30 μL/min. Because FcgammaRIIIa binding differs from theLangmuir 1:1 model, only binding/no binding was determined with thisassay. In a similar manner FcgammaRIa and FcgammaRIIa binding can bedetermined. Results are shown in FIG. 6 , where it follows that byintroduction of the mutations P329G LALA, no more binding toFcgammaRIIIa could be detected.

Assessment of Independent VEGF- and ANG2-Binding to the Anti-VEGF/ANG2Antibodies

Around 3,500 resonance units (RU) of the capturing system (10 μg/mL goatanti-human IgG; GE Healthcare Bio-Sciences AB, Sweden) were coupled on aCM4 chip (GE Healthcare BR-1005-34) at pH 5.0 by using an amine couplingkit supplied by GE Healthcare. The sample and system buffer was PBS-T(10 mM phosphate buffered saline including 0.05% Tween20) pH 7.4. Thetemperature of the flow cell was set to 25° C. and of the sample blockto 12° C. Before capturing, the flow cell was primed with running buffertwice.

The bispecific antibody was captured by injecting a 10 nM solution for60 seconds at a flow of 5 μL/min. Independent binding of each ligand tothe bispecific antibody was analyzed by determining the active bindingcapacity for each ligand, either added sequentially or simultaneously(flow of 30 μL/min):

-   -   1. Injection of human VEGF with a concentration of 200 nM for        180 seconds (identifies the single binding of the antigen).    -   2. Injection of human ANG2 with a concentration of 100 nM for        180 seconds (identifies single binding of the antigen).    -   3. Injection of human VEGF with a concentration of 200 nM for        180 seconds followed by an additional injection of human ANG2        with a concentration of 100 nM for 180 seconds (identifies        binding of ANG2 in the presence of VEGF).    -   4. Injection of human ANG2 with a concentration of 100 nM for        180 seconds followed by an additional injection of human VEGF        with a concentration of 200 nM (identifies binding of VEGF in        the presence of ANG2).    -   5. Co-injection of human VEGF with a concentration of 200 nM and        of human ANG2 with a concentration of 100 nM for 180 seconds        (identifies the binding of VEGF and of ANG2 at the same time).

The surface was regenerated by 60 seconds washing with a 3 M MgCl₂solution at a flow rate of 30 μL/min. Bulk refractive index differenceswere corrected by subtracting the response obtained from a goatanti-human IgG surface.

The bispecific antibody is able to bind both antigens mutualindependently if the resulting final signal of the approaches 3, 4 & 5equals or is similar to the sum of the individual final signals of theapproaches 1 and 2. Results are shown in the Table below, where bothantibodies VEGF/ANG2-0016, VEGF/ANG2-0012 are shown to be able to bindmutual independently to VEGF and ANG2.

Assessment of Simultaneous VEGF- and ANG2-Binding to the Anti-VEGF/ANG2Antibodies

First, around 1,600 resonance units (RU) of VEGF (20 μg/mL) were coupledon a CM4 chip (GE Healthcare BR-1005-34) at pH 5.0 by using an aminecoupling kit supplied by GE Healthcare. The sample and system buffer wasPBS-T (10 mM phosphate buffered saline including 0.05% Tween20) pH 7.4.Flow cell was set to 25° C. and sample block to 12° C. and primed withrunning buffer twice. Second, 50 nM solution of the bispecific antibodywas injected for 180 seconds at a flow of 30 μL/min. Third, hANG2 wasinjected for 180 seconds at a flow of 30 μL/min. The binding response ofhANG2 depends from the amount of the bispecific antibody bound to VEGFand shows simultaneous binding. The surface was regenerated by 60seconds washing with a 0.85% H₃PO₄ solution at a flow rate of 30 μL/min.Simultaneous binding is shown by an additional specific binding signalof hANG2 to the previous VEGF bound anti-VEGF/ANG2 antibodies. For bothbispecific antibodies VEGF/ANG2-0015 and VEGF/ANG2-0016 simultaneousVEGF- and ANG2-binding to the anti-VEGF/ANG2 antibodies could bedetected (data not shown).

TABLE Results: Kinetic affinities to VEGF isoforms from differentspecies VEGF/ ANG2- VEGF/ANG2- VEGF/ANG2- VEGF/ANG2- 0015 - 0016 -0012 - 0201 - apparent apparent apparent apparent affinity affinityaffinity affinity human ≤1 pM ≤1 pM (out of ≤1 pM (out of ≤1 pM (out ofVEGF 121 (out of BIAcore BIAcore BIAcore BIAcore specification)specification) specification) speci- fication) mouse no binding nobinding no binding no binding VEGF 120 rat VEGF 13 nM 14 nM 24 nM 35 nM164

TABLE Results: Solution affinities to ANG2 VEGF/ VEGF/ ANG2- ANG2-VEGF/ANG2- VEGF/ANG2- 0015 0016 0012 0201 KD [nM] KD [nM] KD [nM] KD[nM] human ANG2 8 20 20 n.d. cyno ANG2 5 13 10 n.d. mouse ANG2 8 13 8n.d. rabbit ANG2 4 11 8 n.d.

TABLE Results: Affinity to FcRn of anti-VEGF/ANG2 antibodies VEGF/ VEGF/ANG2- ANG2- 0015 0016 VEGF/ANG2- VEGF/ANG2- [affinity] [affinity] 0012[affinity] 0201 [affinity] human FcRn 0.8 μM no binding no binding 0.8μM cynomolgus 0.9 μM no binding no binding 1.0 μM FcRn mouse FcRn 0.2 μMno binding no binding 0.2 μM

TABLE Results Binding to FcgammaRI - IIIa VEGF/ ANG2- VEGF/ANG2-VEGF/ANG2- VEGF/ANG2- 0015 0016 0012 0201 FcγRIa no binding no bindingbinding binding FcγRIIa no binding no binding no binding bindingFcγRIIIa no binding no binding no binding binding

TABLE Results: Independent binding of VEGF- and ANG2 to anti-VEGF/ANG2antibodies first first VEGF ANG2 Co-injection then then ANG2 + ANG2 VEGFANG2 VEGF VEGF [RUmax] [RUmax] [RUmax] [RUmax] [RUmax] VEGF/ 174 50 211211 211 ANG2- 0016 VEGF/ 143 43 178 177 178 ANG2- 0012

Example 4

Mass Spectrometry

This section describes the characterization of anti-VEGF/ANG2 antibodieswith emphasis on the correct assembly. The expected primary structureswere confirmed by electrospray ionization mass spectrometry (ESI-MS) ofthe deglycosylated, and intact or IdeS-digested (IgG-degrading enzyme ofS. pyogenes) anti-VEGF/ANG2 antibodies. The IdeS-digestion was performedwith 100 μg purified antibody incubated with 2 μg IdeS protease(Fabricator) in 100 mmol/L NaH₂PO₄/Na₂HPO₄, pH 7.1 at 37° C. for 5 h.Subsequently, the antibodies were deglycosylated with N-Glycosidase F,Neuraminidase and O-glycosidase (Roche) in 100 mmol/L NaH₂PO₄/Na₂HPO₄,pH 7.1 at 37° C. for up to 16 hours at a protein concentration of 1mg/mL and subsequently desalted via HPLC on a Sephadex G25 column (GEHealthcare). The total mass was determined via ESI-MS on a maXis 4GUHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMatesource (Advion).

The masses obtained for the IdeS-digested, deglycosylated (Table below),or intact, deglycosylated (Table below) molecules correspond to thepredicted masses deduced from the amino acid sequences for theanti-VEGF/ANG2 antibodies consisting of two different light chainsLC_(ANG2) and LC_(Lucentis), and two different heavy chains HC_(ANG2)and HC_(Lucentis).

TABLE Masses of the deglycosylated and IdeS-digested bispecificanti-VEGF/ANG2 antibodies VEGF/ANG2-0201 (without IHH-AAA mutation) andVEGF/ANG2-0012 (with IHH-AAA mutation) deglycosylated Fc-region F(ab′)₂of the anti- of the anti-VEGF/ANG2 VEGF/ANG2 antibody antibody predictedobserved predicted observed average average average average sample mass[Da] mass [Da] mass [Da] mass [Da] VEGF/ANG2- 99360.8 99360.7 47439.247430.1 0201 VEGF/ANG2- 99360.8 99361.1 47087.7 47082.0 0012

TABLE Masses of the deglycosylated anti-VEGF/ANG2 antibodiesVEGF/ANG2-0016 (with IHH-AAA mutation) and VEGF/ ANG2-0015 (withoutIHH-AAA mutation) deglycosylated anti-VEGF/ANG2 antibody predictedaverage mass observed average mass [Da] [Da] VEGF/ANG2-0016 146156.9146161.2 VEGF/ANG2-0015 146505.3 146509.4

Example 5

FcRn Chromatography

Coupling to Streptavidin Sepharose:

One gram streptavidin sepharose (GE Healthcare) was added to thebiotinylated and dialyzed receptor and incubated for two hours withshaking. The receptor derivatized sepharose was filled in a 1 mL XKcolumn (GE Healthcare).

Chromatography Using the FcRn Affinity Column:

-   Conditions:-   column dimensions: 50 mm×5 mm-   bed height: 5 cm-   loading: 50 μg sample-   equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted to pH    5.5-   elution buffer: 20 mM Tris/HCl, with 150 mM NaCl, adjusted to pH 8.8-   elution: 7.5 CV equilibration buffer, in 30 CV to 100% elution    buffer, 10 CV elution buffer    Human FcRn Affinity Column Chromatography

In the following Table of retention times of anti-VEGF/ANG2 antibodieson affinity columns comprising human FcRn are given. Data were obtainedusing the conditions above.

TABLE Results: retention times of anti-VEGF/ANG2 antibodies antibodyretention time [min] VEGF/ANG2-0015 (without 78.5 IHH-AAA mutation)VEGF/ANG2-0201 (without 78.9 IHH-AAA mutation) VEGF/ANG2-0012 (with IHH-2.7 (void-peak) AAA mutation) VEGF/ANG2-0016 (with IHH- 2.7 (void-peak)AAA mutation)

Example 6

Pharmacokinetic (PK) Properties of Antibodies with IHH-AAA Mutation

PK Data with FcRn Mice Transgenic for Human FcRn

In Life Phase:

The study included female C57BL/6J mice (background); mouse FcRndeficient, but hemizygous transgenic for human FcRn (huFcRn, line276-/tg)

Part 1:

All mice were injected once intravitreally into the right eye with 2μL/animal of the appropriate solution (i.e. 21 μg compound/animal(VEGF/ANG2-0015 (without IHH-AAA mutation)) or 23.6 μg compound/animal(VEGF/ANG2-0016 (with IHH-AAA mutation)).

Mice were allocated to 2 groups with 6 animals each. Blood samples aretaken from group 1 at 2, 24 and 96 hours and from group 2 at 7, 48 and168 hours after dosing.

Injection into the vitreous of the right mouse eye was performed usingthe NanoFil Microsyringe system for nanoliter injection from WorldPrecision Instruments, Inc., Berlin, Germany. Mice were anesthetizedwith 2.5% Isoflurane and for visualization of the mouse eye a Leica MZFL3 microscope with a 40 fold magnification and a ring-light with a LeicaKL 2500 LCD lightning was used. Subsequently, 2 μL of the compound wereinjected using a 35-gauge needle.

Blood was collected via the retrobulbar venous plexus of thecontralateral eye from each animal for the determination of the compoundlevels in serum.

Serum samples of at least 50 μL were obtained from blood after 1 hour atRT by centrifugation (9,300×g) at 4° C. for 3 min. Serum samples werefrozen directly after centrifugation and stored frozen at −80° C. untilanalysis. Treated eyes of the animals of group 1 were isolated 96 hoursafter treatment and of the animals of group 2 168 hours after treatment.Samples were stored frozen at −80° C. until analysis.

Part 2:

All mice were injected once intravenously via the tail vein with 200μL/animal of the appropriate solution (i.e. 21 μg compound/animal(VEGF/ANG2-0015 (without IHH-AAA mutation)) or 23.6 μg compound/animal(VEGF/ANG2-0016 (with IHH-AAA mutation)).

Mice were allocated to 2 groups with 5 animals each. Blood samples aretaken from group 1 at 1, 24 and 96 hours and from group 2 at 7, 48 and168 hours after dosing. Blood was collected via the retrobulbar venousplexus from each animal for the determination of the compound levels inserum.

Serum samples of at least 50 μL were obtained from blood after 1 hour atRT by centrifugation (9,300×g) at 4° C. for 3 min. Serum samples werefrozen directly after centrifugation and stored frozen at −80° C. untilanalysis.

Preparation of Whole Eye Lysates (Mice)

The eye lysates were gained by physico-chemical disintegration of thewhole eye from laboratory animals. For mechanical disruption, each eyewas transferred into a 1.5 mL micro vial with conical bottom. Afterfreeze and thawing, the eyes were washed with 1 mL cell washing bufferonce (Bio-Rad, Bio-Plex Cell Lysis Kit, Cat. No. 171-304011). In thefollowing step, 500 μL of freshly prepared cell lysis buffer were addedand the eyes were grinded using a 1.5 mL tissue grinding pestle (KimbleChase, 1.5 mL pestle, Art. No. 749521-1500). The mixture was then frozenand thawed five times and grinded again. To separate lysate fromremaining tissue the samples were centrifuged for 4 min at 4,500 g.After centrifuging, supernatant was collected and stored at −20° C.until further analysis in the quantification ELISA.

Analysis

The concentrations of the anti-VEGF/ANG2 antibodies in mice serum andeye lysates were determined with an enzyme linked immunosorbent assay(ELISA)

For quantification of anti-VEGF/ANG2 antibodies in mouse serum samplesand eye lysates, a standard solid-phase serial sandwich immunoassay withbiotinylated and digoxigenylated monoclonal antibodies used as captureand detection antibodies was performed. To verify the integrity of thebispecificity of the analyte, the biotinylated capture antibodyrecognizes the VEGF-binding site whereas the digoxigenylated detectionantibody will bind to the ANG2 binding site of the analyte. The boundimmune complex of capture antibody, analyte and detection antibody onthe solid phase of the streptavidin coated micro titer plate (SA-MTP) isthen detected with a horseradish-peroxidase coupled to ananti-digoxigenin antibody. After washing unbound material from theSA-MTP and addition of ABTS-substrate, the gained signal is proportionalto the amount of analyte bound on the solid phase of the SA-MTP.Quantification is then done by converting the measured signals of thesamples into concentrations referring to calibrators analyzed inparallel.

In a first step the SA-MTP was coated with 100 μL/well of biotinylatedcapture antibody solution (mAb<Id<VEGF>>M-2.45.51-IgG-Bi(DDS),anti-idiotypic antibody) with a concentration of 1 μg/mL for one hour at500 rpm on a MTP-shaker. Meanwhile calibrators, QC-samples and sampleswere prepared. Calibrators and QC-samples are diluted to 2% serummatrix; samples were diluted until the signals were within the linearrange of the calibrators.

After coating the SA-MTP with capture antibody, the plate was washedthree times with washing buffer and 300 μL/well. Subsequently, 100μL/well of the calibrators, QC-samples and samples were pipetted on theSA-MTP and incubated again for one hour at 500 rpm. The analyte was nowbound with its VEGF binding site via the capture antibody to the solidphase of the SA-MTP. After incubation and removal of unbound analyte bywashing the plate 100 μL/well of the first detection antibody(mAb<Id-<ANG2>>M-2.6.81-IgG-Dig(XOSu), anti-idiotypic antibody) with aconcentration of 250 ng/mL was added to the SA-MTP. Again, the plate wasincubated for one hour at 500 rpm on a shaker. After washing, 100μL/well of the second detection antibody (pAb<Digoxigenin>S-Fab-POD(poly)) at a concentration of 50 mU/mL was added to the wells of theSA-MTP and the plate was incubated again for one hour at 500 rpm. Aftera final washing step to remove excess detection antibody, 100 μL/wellsubstrate (ABTS) is added. The antibody-enzyme conjugate catalyzes thecolor reaction of the ABTS® substrate. The signal was then measured byELISA reader at 405 nm wavelength (reference wavelength: 490 nm([405/490] nm)).

Pharmacokinetic Evaluation

The pharmacokinetic parameters were calculated by non-compartmentalanalysis, using the pharmacokinetic evaluation program WINNONLIN™(Pharsight), version 5.2.1.

Results:

A) Serum Concentrations

Results for serum concentrations are shown in the following Tables andFIGS. 7B to 7C.

TABLE VEGF/ANG2-0015 (without IHH-AAA mutation): Comparison of serumconcentrations after intravitreal and intravenous application serumconcentration serum concentration after intravitreal after intravenousapplication application ID average conc. [μg/mL] average conc. [μg/mL] 1 h 17.7  2 h 9.8  7 h 10.4 12.1 24 h 6.4 8.3 48 h 6.5 6.9 96 h 3.4 4.1168 h  2.9 2.7

TABLE VEGF/ANG2-0016 (with IHH-AAA mutation): Comparison of serumconcentrations after intravitreal and intravenous application serumconcentration serum concentration after intravitreal after intravenousapplication application ID average conc. [μg/mL] average conc. [μg/mL] 1 h 18.4  2 h 7.0  7 h 8.7 10.0 24 h 2.2 3.3 48 h 1.0 1.0 96 h 0.1 0.1168 h  0.0 0.0

TABLE VEGF/ANG2-0015 (without IHH-AAA mutation) and VEGF/ ANG2-0016(with IHH-AAA mutation): Comparison of serum concentrations afterintravitreal application) VEGF/ANG2-0015 VEGF/ANG2-0016 (without IHH-AAA(with IHH-AAA mutation) mutation) ID average conc. [μg/mL] average conc.[μg/mL]  2 h 9.8 7.0  7 h 10.4 8.7 24 h 6.4 2.2 48 h 6.5 1.0 96 h 3.40.1 168 h  2.9 0.0

TABLE VEGF/ANG2-0015 (without IHH-AAA mutation) and VEGF/ ANG2-0016(with IHH-AAA mutation): Comparison of serum concentrations afterintravenous application VEGF/ANG2-0015 VEGF/ANG2-0016 (without IHH-AAA(with IHH-AAA mutation) mutation) ID average conc. [μg/mL] average conc.[μg/mL]  1 h 17.7 18.4  7 h 12.1 10.0 24 h 8.3 3.3 48 h 6.9 1.0 96 h 4.10.1 168 h  2.7 0.0Results:B) Concentrations in Eye-Lysates of Left and Right Eyes

Results for concentrations in eye lysates are shown in the followingTables and FIGS. 7D to 7E.

TABLE Concentrations of VEGF/ANG2-0015 (without IHH-AAA mutation) in eyelysates after intra vitreal application into right eye mean conc. valuesfrom n = 6 mice ID mean conc. [ng/mL]  96 h left eye 8.7 right eye 46.1168 h left eye 4.3 right eye t 12.9

TABLE Concentrations of VEGF/ANG2-0015 (without IHH-AAA mutation) in eyelysates after intravenous application mean conc. values from n = 5 miceID mean conc. [ng/mL]  96 h left eye 4.2 right eye 7.5 168 h left eye3.4 right eye 6.1

TABLE Concentrations of VEGF/ANG2-0016 (with IHH-AAA mutation) in eyelysates after intra vitreal application into right eye mean conc. valuesfrom n = 5 mice ID mean conc. [ng/mL]  96 h left eye 0.3 right eye 34.5168 h left eye 0.1 right eye 9.0

TABLE Concentrations of VEGF/ANG2-0016 (with IHH-AAA mutation) in eyelysates after intravenous application mean conc. values from n = 5 miceID mean conc. [ng/mL]  96 h left eye 0.0 right eye 0.1 168 h left eye0.0 right eye 0.1Summary of Results:

After intravitreal application the bispecific anti-VEGF/ANG2 antibody asreported herein VEGF/ANG2-0016 (with IHH-AAA mutation) shows similarconcentrations (after 96 and 168 hours) in the eye lysates as comparedto the bispecific anti-VEGF/ANG2 antibody without IHH-AAA mutationVEGF/ANG2-0015.

Also after intravitreal application the bispecific anti-VEGF/ANG2antibody as reported herein VEGF/ANG2-0016 (with IHH-AAA mutation) showsin addition a faster clearance and shorter half-life in the serum ascompared to the bispecific anti-VEGF/ANG2 antibody without IHH-AAAmutation VEGF/ANG2-0015.

Example 7

Mouse Cornea Micropocket Angiogenesis Assay

To test the anti-angiogenic effect, bispecific anti-VEGF/ANG2 antibodywith the respective VEGF binding VH and VL of SEQ ID NO: 20 and 21 andthe ANG2 binding VH and VL of SEQ ID NO: 28 and 29 on VEGF-inducedangiogenesis in vivo, a mouse corneal angiogenesis assay was performed.In this assay a VEGF soaked Nylaflo disc is implanted into a pocket ofthe avascular cornea at a fixed distance to the limbal vessels. Vesselsimmediately grow into the cornea towards the developing VEGF gradient. 8to 10 weeks old female Balb/c mice were purchased from Charles River,Sulzfeld, Germany. The protocol is modified according to the methoddescribed by Rogers, M. S., et al., Nat. Protoc. 2 (2007) 2545-2550.Briefly, micropockets with a width of about 500 μm are prepared under amicroscope at approximately 1 mm from the limbus to the top of thecornea using a surgical blade and sharp tweezers in the anesthetizedmouse. The disc (Nylaflo®, Pall Corporation, Michigan) with a diameterof 0.6 mm is implanted and the surface of the implantation area wassmoothened. Discs are incubated in corresponding growth factor or invehicle for at least 30 min. After 3, 5 and 7 days (or alternativelyonly after 3, 5 or 7 days) eyes are photographed and vascular responseis measured. The assay is quantified by calculating the percentage ofthe area of new vessels per total area of the cornea.

The discs are loaded with 300 ng VEGF or with PBS as a control andimplanted for 7 days. The outgrowth of vessels from the limbus to thedisc is monitored over time on day 3, 5 and/or 7. One day prior to discimplantation, the antibodies are administered intravenously at a dose of10 mg/kg (due to the intravenous application the serum-stableVEGF/ANG2-0015 (without IHH-AAA mutation) which only differs fromVEGF/ANG2-0016 by the IHH-AAA mutation and has the same VEGF and ANG2binding VHs and VLs to mediate efficacy, is used as surrogate) fortesting the anti-angiogenic effect on VEGF-induced angiogenesis in vivo.Animals in the control group receive vehicle. The application volume is10 mL/kg.

Example 8

Pharmacokinetic (PK) Properties of Antibodies with HHY-AAA Mutation

PK Data with FcRn Mice Transgenic for Human FcRn

In Life Phase:

The study included female C57BL/6J mice (background); mouse FcRndeficient, but hemizygous transgenic for human FcRn (huFcRn, line276-/tg)

Part 1:

All mice were injected once intravitreally into the right eye with theappropriate solution of IGF-1R 0033, IGF-1R 0035, IGF-1R 0045 (i.e. 22.2μg compound/animal of IGF-1R 0033, 24.4 μg compound/animal IGF-1R 0035,32.0 μg compound/animal IGF-1R and 32.0 μg compound/animal of IGF-1R0045).

Thirteen mice were allocated to 2 groups with 6 and 7, respectively,animals each. Blood samples are taken from group 1 at 2, 24 and 96 hoursand from group 2 at 7, 48 and 168 hours after dosing.

Injection into the vitreous of the right mouse eye was performed byusing the NanoFil Microsyringe system for nanoliter injection from WorldPrecision Instruments, Inc., Berlin, Germany. Mice were anesthetizedwith 2.5% Isoflurane and for visualization of the mouse eye a Leica MZFL3 microscope with a 40 fold magnification and a ring-light with a LeicaKL 2500 LCD lightning was used. Subsequently, 2 μL of the compound wereinjected using a 35-gauge needle.

Blood was collected via the retrobulbar venous plexus of thecontralateral eye from each animal for the determination of the compoundlevels in serum.

Serum samples of at least 50 μL were obtained from blood after 1 hour atRT by centrifugation (9,300×g) at 4° C. for 3 min. Serum samples werefrozen directly after centrifugation and stored frozen at −80° C. untilanalysis. Treated eyes of the animals of group 1 were isolated 96 hoursafter treatment and of the animals of group 2 168 hours after treatment.Samples were stored frozen at −80° C. until analysis.

Part 2:

All mice were injected once intravenously via the tail vein with theappropriate solution of IGF-1R 0033, IGF-1R 0035, IGF-1R 0045 (i.e. 22.2μg compound/animal of IGF-1R 0033, 24.4 μg compound/animal IGF-1R 0035,32.0 μg compound/animal IGF-1R and 32.0 μg compound/animal of IGF-1R0045).

Twelve mice were allocated to 2 groups with 6 animals each. Bloodsamples are taken from group 1 at 1, 24 and 96 hours and from group 2 at7, 48 and 168 hours after dosing. Blood was collected via theretrobulbar venous plexus from each animal for the determination of thecompound levels in serum.

Serum samples of at least 50 μL were obtained from blood after 1 hour atRT by centrifugation (9,300×g) at 4° C. for 3 min. Serum samples werefrozen directly after centrifugation and stored frozen at −80° C. untilanalysis.

Preparation of Cell Lysis Buffer

Carefully mix 100 μL factor 1, 50 μL factor 2 and 24.73 mL Cell Lysisbuffer (all from Bio-Rad, Bio-Plex Cell Lysis Kit, Cat. No. 171-304011)and add 125 μL PMSF solution (174.4 mg phenylmethylsulfonylfluoridediluted in 2.0 mL DMSO).

Preparation of Whole Eye Lysates (Mice)

The eye lysates were gained by physico-chemical disintegration of thewhole eye from laboratory animals. For mechanical disruption each eyewas transferred into a 1.5 mL micro vial with conical bottom. Afterthawing, the eyes were washed with 1 mL cell washing buffer once(Bio-Rad, Bio-Plex Cell Lysis Kit, Cat. No. 171-304011). In thefollowing step 500 μL of freshly prepared cell lysis buffer were addedand the eyes were grinded using a 1.5 mL tissue grinding pestle (VWRInt., Art. No. 431-0098). The mixture was then frozen and thawed fivetimes and grinded again. To separate lysate from remaining tissue thesamples were centrifuged for 4 min. at 4500×g. After centrifuging thesupernatant was collected and stored at −20° C. until further analysisin the quantification ELISA

Analysis (Serum)

For quantification of antibodies in mouse serum sample, a standardsolid-phase serial sandwich immunoassay with biotinylated anddigoxigenated monoclonal antibodies used as capture and detectionantibodies is performed. Serum accounts for about 50% of the full bloodsample volume.

More detailed, concentrations of the antibodies in mouse serum sampleswere determined by a human-IgG (Fab) specific enzyme linkedimmunosorbent assay. Streptavidin coated microtiter plates wereincubated with the biotinylated anti-human Fab(kappa) monoclonalantibody M-1.7.10-IgG as capture antibody diluted in assay buffer forone hour at room temperature with agitation. After washing three timeswith phosphate-buffered saline-polysorbate 20 (Tween20), serum samplesat various dilutions were added followed by second incubation for onehour at room temperature. After three repeated washings bound antibodywas detected by subsequent incubation with the anti-human Fab(CH1)monoclonal antibody M-1.19.31-IgG conjugated to digoxigenin, followed byan anti-digoxigenin antibody conjugated to horseradish peroxidase (HRP).ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); RocheDiagnostics GmbH, Mannheim, Germany) was used as HRP substrate to form acolored reaction product. Absorbance of the resulting reaction productwas read at 405 nm (ABTS; reference wavelength: 490 nm).

All samples, positive and negative control samples were analyzed inreplicates and calibrated against an antibody standard provided.

Analysis (Eye Lysate)

The concentrations of the analytes in mouse eye lysate samples weredetermined using a qualified electro-chemiluminescence immunoassay(ECLIA) method based on the ELECSYS® instrument platform (RocheDiagnostics GmbH, Mannheim, Germany) under non-GLP conditions.

The undiluted supernatant (eye lysates) was incubated with capture anddetection molecules for 9 min. at 37° C. Biotinylatedanti-human-Fab(kappa) monoclonal antibody M-1.7.10-IgG was used ascapture molecule and a ruthenium(II)tris(bispyridyl)₃ ²⁺ labeledanti-human-Fab(CH1) monoclonal antibody M-1.19.31-IgG was used fordetection. Streptavidin-coated magnetic microparticles were added andincubated for additional 9 min. at 37° C. to allow binding of preformedimmune complexes due to biotin-streptavidin interactions. Themicroparticles were magnetically captured on an electrode and achemiluminescent signal generated using the co-reactant tripropyl amine(TPA). The gained signal was measured by a photomultiplier detector.

TABLE Standard chart IGF-1R 0033 standard deviation serum- concentrationsignal mean signal conc. recovery [ng/mL] counts counts [ng/mL] [%]standard sample 9 0 1038 46 — — standard sample 8 0.686 2682 105 0.67598 standard sample 7 2.06 6275 791 2.06 100 standard sample 6 6.17 15907316 6.23 101 standard sample 5 18.5 45455 1238 18.8 102 standard sample4 55.6 133940 949 55.7 100 standard sample 3 167 388069 2929 165 99standard sample 2 500 1129804 16777 503 101 standard sample 1 15002956965 60287 1499 100

TABLE Standard chart IGF-1R 0035 standard deviation serum- concentrationsignal mean signal conc. recovery [ng/mL] counts counts [ng/mL] [%]standard sample 9 0 1024 63 — — standard sample 8 0.686 2817 38 0.681 99standard sample 7 2.06 6451 39 2.08 101 standard sample 6 6.17 17100 3196.13 99 standard sample 5 18.5 49693 713 18.6 100 standard sample 4 55.6146746 2575 56.1 101 standard sample 3 167 423597 5068 165 99 standardsample 2 500 1224244 11655 502 100 standard sample 1 1500 3144901 445361499 100

TABLE Standard chart IGF-1R 0045 standard deviation serum- concentrationsignal mean signal conc. recovery [ng/mL] counts counts [ng/mL] [%]standard sample 9 0 1339 545 — — standard sample 8 0.686 3108 61 0.62291 standard sample 7 2.06 7032 189 1.93 94 standard sample 6 6.17 19175750 6.10 99 standard sample 5 18.5 55526 823 18.7 101 standard sample 455.6 158591 5412 55.7 100 standard sample 3 167 456316 28759 167 100standard sample 2 500 1274801 47532 499 100 standard sample 1 15003280452 239523 1501 100Results:A) Serum Concentrations

Results for serum concentrations are shown in the following Tables andFIG. 17 .

TABLE IGF-1R 0033 (without HHY-AAA mutation): Comparison of serumconcentrations after intravitreal and intravenous application serumconcentration serum concentration after intravitreal after intravenousapplication application ID average conc. [μg/mL] average conc. [μg/mL] 1 h n.d. 34.7  2 h 5.9 n.d.  7 h 11.1 24.7 24 h 4.4 13.6 48 h 7.8 12.696 h 2.1 8.9 168 h  2.9 6.2 (n.d. = not determined)

TABLE IGF-1R 0035 (with HHY-AAA mutation in one Fc-region polypeptide):Comparison of serum concentrations after intravitreal and intravenousapplication serum concentration serum concentration after intravitrealafter intravenous application application ID average conc. [μg/mL]average conc. [μg/mL]  1 h n.d. 24.5  2 h 7.3 n.d.  7 h 7.9 16.1 24 h2.3 5.7 48 h 1.7 2.9 96 h 0.3 0.6 168 h  0.1 0.2

TABLE IGF-1R 0045 (with HHY-AAA mutation in both Fc-regionpolypeptides): Comparison of serum concentrations after intravitreal andintravenous application (BLQ = below limit of quantitation) serumconcentration serum concentration after intravitreal after intravenousapplication application ID average conc. [μg/mL] average conc. [μg/mL] 1 h n.d. 40.5  2 h 13.2 n.d.  7 h 9.6 21.7 24 h 2.2 5.1 48 h 0.9 0.7 96h 0.05 0.03 168 h  0.01 BLQ

TABLE Comparison of serum concentrations after intravenous applicationof antibodies IGF-1R 0033, 0035 and 0045 normalized to 1 μg appliedantibody IGF-1R 0033 IGF-1R 0035 IGF-1R 0045 ID average conc. [ng/mL/μgapplied antibody]  1 h 1564 1006 1266  7 h 1114 659 679 24 h 613 234 16048 h 569 118 21 96 h 399 26 1 168 h  280 7 0Results:B) Concentrations in Eye-Lysates of Left and Right Eyes

Results for concentrations in eye lysates are shown in the followingTables and FIGS. 18 to 20 .

TABLE Concentrations of IGF-1R 0033 (without HHY-AAA mutation) in eyelysates after intravitreal application into the right eye mean conc.values from n = 7 (96 h) and n = 6 (196 h) mice ID mean conc. [ng/mL] 96 h left eye 3.3 right eye 99.5 168 h left eye 5.2 right eye 144.9

TABLE Concentrations of IGF-1R 0033 (without HHY-AAA mutation) in eyelysates after intravenous application (BLQ = below limit ofquantitation) mean conc. values from n = 5 (96 h) and n = 6 (196 h) miceID mean conc. [ng/mL]  96 h left eye 12.7 right eye 8.5 168 h left eye9.7 right eye BLQ

TABLE Concentrations of IGF-1R 0035 (with the HHY-AAA mutation in oneFc-region polypeptide) in eye lysates after intravitreal applicationinto the right eye mean conc. values from n = 6 mice ID mean conc.[ng/mL]  96 h left eye 1.1 right eye 169.2 168 h left eye 0.3 right eye114.7

TABLE Concentrations of IGF-1R 0035 (with the HHY-AAA mutation in oneFc- region polypeptide) in eye lysates after intravenous application(BLQ = below limit of quantitation) mean conc. values from n = 6 mice IDmean conc. [ng/mL]  96 h left eye 3.7 right eye 1.7 168 h left eye 1.4right eye 0.3

TABLE Concentrations of IGF-1R 0045 (with the HHY-AAA mutation in bothFc-region polypeptides) in eye lysates after intravitreal applicationinto the right eye mean conc. values from n = 6 mice ID mean conc.[ng/mL]  96 h left eye 1.4 right eye 322.6 168 h left eye 1.4 right eye156.8

TABLE Concentrations of IGF-1R 0045 (with the HHY-AAA mutation in bothFc-region polypeptides) in eye lysates after intravenous application(BLQ = below limit of quantitation) mean conc. values from n = 6 (96 h)and n = 5 (196 h) mice ID mean conc. [ng/mL]  96 h left eye 3.6 righteye 1.3 168 h left eye 0.8 right eye 0.4

TABLE Concentrations of IGF-1R 0033, 0035 and 0045 in eye lysates afterintravitreal application into the right eye normalized to 1 μg appliedantibody IGF-1R 0033 IGF-1R 0035 IGF-1R 0045 ID mean conc. [ng/mL]  96 hleft eye 0.15 0.05 0.04 right eye 4.48 6.93 10.08 168 h left eye 0.240.01 0.04 right eye 6.53 4.70 4.90Summary of Results:

After intravitreal application, the anti-IGF-1R antibodies 0035 and 0045as reported herein (with one sided or both sided HHY-AAA mutation) showssimilar concentrations (after 96 and 168 hours) in the eye lysates ascompared to the anti-IGF-1R antibody without HHY-AAA mutation (IGF-1R0033).

Also after intravitreal application the anti-IGF-1R antibodies 0035 and0045 as reported herein (with one sided or both sided HHY-AAA mutation)shows in addition a faster clearance and shorter half-life in the serumas compared to the anti-IGF-1R antibody without HHY-AAA mutation (IGF-1R0033).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. An antibody comprising a first polypeptide and a second polypeptide each comprising, in N-terminal to C-terminal direction, at least a portion of an immunoglobulin hinge region, which comprises one or more cysteine residues, an immunoglobulin CH2-domain and an immunoglobulin CH3-domain, wherein: the first and/or the second polypeptide comprises the mutation Y436A (numbering according to Kabat), the antibody is a full length antibody with a human IgG1 Fc-region (numbering according to Kabat), and the antibody is a bispecific antibody.
 2. The antibody according to claim 1, wherein the antibody is a monoclonal antibody.
 3. The antibody according to claim 1, wherein the antibody is a human, humanized or chimeric antibody.
 4. The antibody according to claim 1, wherein the antibody is a bivalent antibody.
 5. The antibody according to claim 1, wherein the antibody s specifically binds to Staphylococcal protein A.
 6. The antibody according to claim 1, wherein i) the first Fc-region polypeptide is selected from the group comprising human IgG1 Fc-region polypeptide, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, human IgG1 Fc-region polypeptide with the mutations Y349C, T366S, L368A, Y407V, human IgG1 Fc-region polypeptide with the mutations S354C, T366S, L368A, Y407V, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, Y349C, T366S, L368A, Y407V, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, S354C, T366S, L368A, Y407V, human IgG1 Fc-region polypeptide with the mutations P329G, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G, human IgG1 Fc-region polypeptide with the mutations P329G, Y349C, T366S, L368A, Y407V, human IgG1 Fc-region polypeptide with the mutations P329G, S354C, T366S, L368A, Y407V, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G, Y349C, T366S, L368A, Y407V, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G, S354C, T366S, L368A, Y407V, human IgG1 with the mutations K392D, and and ii) the second Fc-region polypeptide is selected from the group comprising human IgG1 Fc-region polypeptide, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, human IgG1 Fc-region polypeptide with the mutations S354C, T366W, human IgG1 Fc-region polypeptide with the mutations Y349C, T366W, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, S354C, T366W, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, Y349C, T366W, human IgG1 Fc-region polypeptide with the mutations P329G, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G, human IgG1 Fc-region polypeptide with the mutations P329G, S354C, T366W, human IgG1 Fc-region polypeptide with the mutations P329G, Y349C, T366W, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G, S354C, T366W, human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G, Y349C, T366W, and human IgG1 with the mutations D399K, D356K, and/or E357K.
 7. The antibody according to claim 1, wherein i) the first Fc-region polypeptide is a human IgG1 Fc-region polypeptide and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide, or ii) the first Fc-region polypeptide is a human IgG1 Fc-region polypeptide with the mutations L234A, L235A and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide with the mutations L234A, L235A, or iii) the first Fc-region polypeptide is a human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G, or iv) the first Fc-region polypeptide is a human IgG1 Fc-region polypeptide with the mutations L234A, L235A, S354C, T366W and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide with the mutations L234A, L235A, Y349C, T366S, L368A, Y407V, or v) the first Fc-region polypeptide is a human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G, S354C, T366W and the second Fc-region polypeptide is a human IgG1 Fc-region polypeptide with the mutations L234A, L235A, P329G, Y349C, T366S, L368A, Y407V.
 8. A pharmaceutical formulation comprising the antibody according to claim 1 and a pharmaceutically acceptable carrier. 